RAF gene fusions

ABSTRACT

The present disclosure relates to compositions and methods for cancer diagnosis, research and therapy, including but not limited to, cancer markers. In particular, the present disclosure relates to RAF gene fusions as diagnostic markers and clinical targets for cancer.

This application is a divisional of U.S. patent application Ser. No.13/300,063, filed Nov. 18, 2011, which claims priority to U.S.provisional patent application 61/415,495, filed Nov. 19, 2010, each ofwhich is herein incorporated by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under CA132874,CA069568, CA111275 and DA021519 awarded by the National Institutes ofHealth and W81XWH-09-2-0013 awarded by the Army/MRMC. The government hascertain rights in the invention.

FIELD OF THE INVENTION

The present disclosure relates to compositions and methods for cancerdiagnosis, research and therapy, including but not limited to, cancermarkers. In particular, the present disclosure relates to RAF genefusions as diagnostic markers and clinical targets for cancer.

BACKGROUND OF THE INVENTION

A central aim in cancer research is to identify altered genes that arecausally implicated in oncogenesis. Several types of somatic mutationshave been identified including base substitutions, insertions,deletions, translocations, and chromosomal gains and losses, all ofwhich result in altered activity of an oncogene or tumor suppressorgene. First hypothesized in the early 1900's, there is now compellingevidence for a causal role for chromosomal rearrangements in cancer(Rowley, Nat Rev Cancer 1: 245 (2001)). Recurrent chromosomalaberrations were thought to be primarily characteristic of leukemias,lymphomas, and sarcomas. Epithelial tumors (carcinomas), which are muchmore common and contribute to a relatively large fraction of themorbidity and mortality associated with human cancer, comprise less than1% of the known, disease-specific chromosomal rearrangements (Mitelman,Mutat Res 462: 247 (2000)). While hematological malignancies are oftencharacterized by balanced, disease-specific chromosomal rearrangements,most solid tumors have a plethora of non-specific chromosomalaberrations. It is thought that the karyotypic complexity of solidtumors is due to secondary alterations acquired through cancer evolutionor progression.

Two primary mechanisms of chromosomal rearrangements have beendescribed. In one mechanism, promoter/enhancer elements of one gene arerearranged adjacent to a proto-oncogene, thus causing altered expressionof an oncogenic protein. This type of translocation is exemplified bythe apposition of immunoglobulin (IG) and T-cell receptor (TCR) genes toMYC leading to activation of this oncogene in B- and T-cellmalignancies, respectively (Rabbitts, Nature 372: 143 (1994)). In thesecond mechanism, rearrangement results in the fusion of two genes,which produces a fusion protein that may have a new function or alteredactivity. The prototypic example of this translocation is the BCR-ABLgene fusion in chronic myelogenous leukemia (CML) (Rowley, Nature 243:290 (1973); de Klein et al., Nature 300: 765 (1982)). Importantly, thisfinding led to the rational development of imatinib mesylate (Gleevec),which successfully targets the BCR-ABL kinase (Deininger et al., Blood105: 2640 (2005)). Thus, identifying recurrent gene rearrangements incommon epithelial tumors may have profound implications for cancer drugdiscovery efforts as well as patient treatment.

SUMMARY OF THE INVENTION

The present disclosure relates to compositions and methods for cancerdiagnosis, screening, research and therapy, including but not limitedto, cancer markers. In particular, the present disclosure relates to RAFgene fusions as diagnostic markers and clinical targets for cancer.

Embodiments of the present disclosure provide a method for identifying aneoplastic cell (e.g., gastric cancer or melanoma) in a patientcomprising: exposing a patient sample comprising a cell, or a secretionthereof to a detection reagent; and detecting the presence or absence inthe sample of a gene fusion having a 5′ portion from a transcriptionalregulatory region of, for example, an SLC45A3, RAF family member, AGTRAPor ESRP1 gene and a 3′ portion from a RAF family member gene or an ESRP1gene, wherein detecting the presence in the sample of the gene fusionidentifies cancer (e.g., gastric cancer or melanoma) in the patient. Insome embodiments, the transcriptional regulatory region of the SLC45A3,RAF family member, AGTRAP or ESRP1 gene comprises a promoter region ofthe gene. In some embodiments, the detecting comprises detectingchromosomal rearrangements of genomic DNA having a 5′ DNA portion fromthe transcriptional regulatory region of the SLC45A3, RAF family member,AGTRAP or ESRP1 gene and a 3′ DNA portion from the RAF family membergene or the ESPR1 gene. In other embodiments, the detecting comprisesdetecting chimeric mRNA transcripts having a 5′ RNA portion transcribedfrom the transcriptional regulatory region of the SLC45A3, RAF familymember, AGTRAP or ESRP1 gene and a 3′ RNA portion transcribed from a RAFfamily member gene or an ESRP1 gene. In some embodiments, the sample is,for example, tissue, blood, plasma, serum, urine, urine supernatant,urine cell pellet or cells (e.g., gastric or skin cells or a fecalsample). In some embodiments, the RAF family member gene is BRAF orRAF1. In some embodiments, the method further comprises the step ofdetecting the level of expression of the gene fusion in the sample,wherein detecting an enhanced level of expression of the gene fusion inthe patient sample relative to the level of expression of the genefusion in a normal sample (e.g., relative to the level in normal cells,increase or decrease in level relative to a prior time point, increaseor decrease relative to a pre-established threshold level, etc.)indicates the presence of a neoplastic prostate cell or a cellpredisposed to the onset of a neoplastic state in the sample.

Further embodiments provide the step of determining a treatment courseof action based on the presence or absence of the gene fusion. Forexample, in some embodiments, the treatment course of action comprisesadministration of a RAF pathway inhibitor (e.g., BAY43-9006, PLX4720, AZ628, GCD 0879 or PLX4032) when the gene fusion is present in the sample.

Additional embodiments of the present disclosure provide compositions,kits or systems comprising at least one of the following: (a) anoligonucleotide probe comprising a sequence that hybridizes to ajunction of a chimeric genomic DNA or chimeric mRNA in which a 5′portion of the chimeric genomic DNA or chimeric mRNA is from atranscriptional regulatory region of an AGTRAP gene and a 3′ portion ofthe chimeric genomic DNA or chimeric mRNA is from a RAF family membergene; (b) a first oligonucleotide probe comprising a sequence thathybridizes to a 5′ portion of a chimeric genomic DNA or chimeric mRNAfrom a transcriptional regulatory region of an AGTRAP gene and a secondoligonucleotide probe comprising a sequence that hybridizes to a 3′portion of the chimeric genomic DNA or chimeric mRNA from a RAF familymember gene; (c) a first amplification oligonucleotide comprising asequence that hybridizes to a 5′ portion of a chimeric genomic DNA orchimeric mRNA from a transcriptional regulatory region of an AGTRAP geneand a second amplification oligonucleotide comprising a sequence thathybridizes to a 3′ portion of the chimeric genomic DNA or chimeric mRNAfrom a RAF family member gene; (d) an oligonucleotide probe comprising asequence that hybridizes to a junction of a chimeric genomic DNA orchimeric mRNA in which a 5′ portion of the chimeric genomic DNA orchimeric mRNA is from a transcriptional regulatory region of a RAFfamily member gene and a 3′ portion of the chimeric genomic DNA orchimeric mRNA is from an ESRP1 member gene; (e) a first oligonucleotideprobe comprising a sequence that hybridizes to a 5′ portion of achimeric genomic DNA or chimeric mRNA from a transcriptional regulatoryregion of a RAF family member gene and a second oligonucleotide probecomprising a sequence that hybridizes to a 3′ portion of the chimericgenomic DNA or chimeric mRNA from an ESRP1 gene; or (f) a firstamplification oligonucleotide comprising a sequence that hybridizes to a5′ portion of a chimeric genomic DNA or chimeric mRNA from atranscriptional regulatory region of a RAF family member gene and asecond amplification oligonucleotide comprising a sequence thathybridizes to a 3′ portion of the chimeric genomic DNA or chimeric mRNAfrom an ESRP1 gene. In some embodiments, the RAF family member gene isBRAF or RAF1.

Additional embodiments of the present disclosure are provided in thedescription and examples below.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the identification of the SLC45A3-BRAF and ESRP1-RAF1 genefusions in prostate cancer by paired-end transcriptome sequencing. (a)Histograms of gene fusion nomination scores in clinically localizedprostate tumor samples PCA1, PCA2, PCA3 and PCA17 harboringAX747630-ETV1, TMPRSS2-ERG, SLC45A3-BRAF, ESRP1-RAF1 and RAF1-ESR1,respectively, and a gastric cancer sample, GCT15, harboring AGTRAP-BRAF.(b) Schematic representation of reliable paired-end reads supporting theinterchromosomal gene fusion between SLC45A3 and BRAF. (c,d) As in b,except showing the fusions between ESRP1 and RAF1, resulting inreciprocal fusion genes ESRP1-RAF1 and RAF1-ESRP1. (e) As in b, exceptshowing the fusion between AGTRAP and BRAF.

FIG. 2 shows experimental validation of the SLC45A3-BRAF, ESRP1-RAF1 andRAF1-ESRP1 and AGTRAP-BRAF gene fusions in the prostate tumor samplesand prostate cancer cell lines (DU145, VCaP, LnCaP and 22RV1) and RWPEas negative controls. (a-c) Expression of SLC45A3-BRAF gene fusion inPCA3 (a), ESRP1-RAF1 and RAF1-ESRP1 fusions in PCA17 (b) and AGTRAP-BRAFfusion in GCT15 (c) tumors are validated by qRTPCR by normalizingagainst glyceraldehyde 6-phosphate dehydrogenase (GAPDH) values in eachsample. (d) FISH validation of SLC45A3-BRAF (left) and ESRP1-RAF1(right) gene fusions in PCA3 and PCA17 tumors, respectively. (e) FISHvalidation of the BRAF rearrangement in GCT15 tumor (left and BRAF 5′deletion (right). (f) FISH validation of the BRAF rearrangement inmelanoma case MEL23 (left) and RAFT rearrangement in melanoma case MEL24(right). (g) Expression of the 120-kDa ESRP1-RAF1 fusion protein in theindex case PCA17. (h) Expression of a 70-kDa AGTRAP-BRAF fusion proteinin case GCT15.

FIG. 3 shows oncogenic properties of SLC45A3-BRAF and ESRP1-RAF1 genefusions. (a) Foci formation by SLC45A3-BRAF, BRAFV600E and vectorcontrol (pDEST40 and pBABE) constructs in NIH3T3 cells. (b) Tumor growthin nude mice implanted with NIH3T3 cells overexpressing SLC45A3-BRAF orpDEST40 vector control. (c,d) Cell proliferation assay using RWPE cellsoverexpressing SLC45A3-BRAF (c) and ESRP1-RAF1 (d) gene fusions.

FIG. 4 shows that RAF and MEK inhibitors block SLC45A3-BRAF orESRP1-RAF1 gene fusion-mediated oncogenic phenotypes. (a,b)SLC45A3-BRAF- or ESRP1-RAF1-mediated cell invasion in RWPE prostatecells is sensitive to sorafenib (10 μM) or the MEK inhibitor U0126 (1 or10 μM). (a) Crystal violet staining of cells after invasion throughMatrigel. (b) Quantification of cells by absorbance. (c,d)Photomicrographs (c) or quantification (d) of SLC45A3-BRAF- orESRP1-RAF1-induced anchorage-independent colony growth in soft agar,which was sensitive to sorafenib or U0126. (e) Evaluation of thedownstream signaling pathways activated by the SLC45A3-BRAF orESRP1-RAF1 gene fusions in RWPE prostate cells.

FIG. 5 shows exon and protein domain structure of BRAF and RAF1 wildtype and fusion gene constructs. a, Schematic diagram showing the exonstructure of wild type SLC45A3 and BRAF and SLC45A3-BRAF fusion gene inthe top panel. b. Full length SLC45A3-BRAF fusion transcript (2017 bp)containing the first un-translated exon of SLC45A3 and exon 8 to thelast exon of BRAF was cloned into pDEST40 vector. c, Schematic diagramshowing the exon structure of wild type ESRP1 and RAF1 genes andESRP1-RAF1 (left) and RAF1-ESRP1 (right) reciprocal fusion transcriptsin the top panel.

FIG. 6 shows that ESRP1, the 5′ fusion partner of RAF1 is not regulatedby androgen.

FIG. 7 shows RNA-seq exon coverage and qRT-PCR validation of BRAF exonsin normal, metastatic prostate samples and index case (PCA3). a. Exonsare shown at the bottom in alternating shades of grey. b. qRT-PCR usingexon spanning primers showing high level expression of BRAF exons 8-18relative to the exons 1-7 in PCA3.

FIG. 8 shows genomic organization and FISH validation of BRAF and RAF1gene rearrangement. a, Schematic diagrams showing the genomic locationof SLC45A3 (left) and BRAF (right) genes on chromosome 1q32.1 and 7q34respectively. b, Schematic diagrams showing the genomic location ofESRP1 (right) and RAF1 (left) genes on chromosome 8q22.1 and 3p25.1respectively.

FIG. 9 shows validation of expression constructs by qRT-PCR and westernblot analysis. a. SLC45A3-BRAF expression constructs with N-terminusFlag tag and C-terminus V5 tag were transfected in HEK293 cells. b.Stable expression of BRAF EX8-Stop and SLC45A3-BRAF fusion construct inRWPE cells was validated by qRT-PCR and western blot analysis using BRAFspecific antibody. c. Stable expression of ESRP1-RAF1 fusion constructin RWPE cells was validated by qRT-PCR (left panel) and western blotanalysis (right panel) using RAF1 specific antibody.

FIG. 10 shows a, Comparison of the foci frequencies of NIH3T3 cellsexpressing fusion transcript SLC45A3-BRAF, BRAF Ex8-Stop and BRAFEx10-Stop and pDEST40 vector. b, Stable RWPE cells over-expressingSLC45A3-BRAF form small tumors in Balb C nu/nu mice.

FIG. 11 shows down-regulation of genes involved in the MEK pathway afterU0126 treatment. a, Stable RWPE cells expressing SLC45A3-BRAF orESRP1-RAF1 showed increase in DUSP6 or SPRY2 mRNA expression as comparedto pDEST40 vector. b, c, MEK inhibitor (U0126, 10 mM) treatment for 2hours in Keratinocyte-supplement free media significantly decreasesexpression of these genes in RWPE cells expressing SLC45A3-BRAF orESRP1-RAF1.

DEFINITIONS

Unless defined otherwise, all terms of art, notations and otherscientific terms or terminology used herein have the same meaning as iscommonly understood by one of ordinary skill in the art to which thisdisclosure belongs. Many of the techniques and procedures described orreferenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art. As appropriate,procedures involving the use of commercially available kits and reagentsare generally carried out in accordance with manufacturer definedprotocols and/or parameters unless otherwise noted. All patents,applications, published applications and other publications referred toherein are incorporated by reference in their entirety. If a definitionset forth in this section is contrary to or otherwise inconsistent witha definition set forth in the patents, applications, publishedapplications, and other publications that are herein incorporated byreference, the definition set forth in this section prevails over thedefinition that is incorporated herein by reference.

As used herein, “a” or “an” means “at least one” or “one or more.”

As used herein, the term “gene fusion” refers to a chimeric genomic DNA,a chimeric messenger RNA, a truncated protein or a chimeric proteinresulting from the fusion of at least a portion of a first gene to atleast a portion of a second gene. The gene fusion need not includeentire genes or exons of genes.

As used herein, the term “gene upregulated in cancer” refers to a genethat is expressed (e.g., mRNA or protein expression) at a higher levelin cancer (e.g., prostate cancer) relative to the level in other tissue.In this context, “other tissue” may refer to, for example, tissues fromdifferent organs in the same subject or to normal tissues of the same ordifferent type. In some embodiments, genes upregulated in cancer areexpressed at a level between at least 10% to 300% higher than the levelof expression in other tissue. For example, genes upregulated in cancerare frequently expressed at a level preferably at least 25%, at least50%, at least 100%, at least 200%, or at least 300% higher than thelevel of expression in other tissue. In some embodiments, genesupregulated in prostate cancer are “androgen regulated genes.”

As used herein, the term “gene upregulated in prostate tissue” refers toa gene that is expressed (e.g., mRNA or protein expression) at a higherlevel in prostate tissue relative to the level in other tissue. In someembodiments, genes upregulated in prostate tissue are expressed at alevel between at least 10% to 300%. For example, genes upregulated incancer are frequently expressed at a level preferably at least 25%, atleast 50%, at least 100%, at least 200%, or at least 300% higher thanthe level of expression in other tissues. In some embodiments, genesupregulated in prostate tissue are exclusively expressed in prostatetissue.

As used herein, the term “transcriptional regulatory region” refers tothe region of a gene comprising sequences that modulate (e.g.,upregulate or downregulate) expression of the gene. In some embodiments,the transcriptional regulatory region of a gene comprises a non-codingupstream sequence of a gene, also called the 5′ untranslated region(5′UTR). In other embodiments, the transcriptional regulatory regioncontains sequences located within the coding region of a gene or withinan intron (e.g., enhancers).

As used herein, the term “androgen regulated gene” refers to a gene orportion of a gene whose expression is induced or repressed by anandrogen (e.g., testosterone). The promoter region of an androgenregulated gene may contain an “androgen response element” that interactswith androgens or androgen signaling molecules (e.g., downstreamsignaling molecules).

As used herein, the terms “detect”, “detecting” or “detection” maydescribe either the general act of discovering or discerning or thespecific observation of a detectably labeled composition.

As used herein, the term “stage of cancer” refers to a qualitative orquantitative assessment of the level of advancement of a cancer.Criteria used to determine the stage of a cancer include, but are notlimited to, the size of the tumor and the extent of metastases (e.g.,localized or distant).

As used herein, the term “nucleic acid molecule” refers to any nucleicacid containing molecule, including but not limited to, DNA or RNA. Theterm encompasses sequences that include any of the known base analogs ofDNA and RNA including, but not limited to, 4-acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatcomprises coding sequences necessary for the production of apolypeptide, precursor, or RNA (e.g., rRNA, tRNA). The polypeptide canbe encoded by a full length coding sequence or by any portion of thecoding sequence so long as the desired activity or functional properties(e.g., enzymatic activity, ligand binding, signal transduction,immunogenicity, etc.) of the full-length or fragment are retained. Theterm also encompasses the coding region of a structural gene and thesequences located adjacent to the coding region on both the 5′ and 3′ends for a distance of about 1 kb or more on either end such that thegene corresponds to the length of the full-length mRNA. Sequenceslocated 5′ of the coding region and present on the mRNA are referred toas 5′ non-translated sequences. Sequences located 3′ or downstream ofthe coding region and present on the mRNA are referred to as 3′non-translated sequences. The term “gene” encompasses both cDNA andgenomic forms of a gene. A genomic form or clone of a gene contains thecoding region interrupted with non-coding sequences termed “introns” or“intervening regions” or “intervening sequences.” Introns are segmentsof a gene that are transcribed into nuclear RNA (hnRNA); introns maycontain regulatory elements such as enhancers. Introns are removed or“spliced out” from the nuclear or primary transcript; introns thereforeare absent in the messenger RNA (mRNA) transcript. The mRNA functionsduring translation to specify the sequence or order of amino acids in anascent polypeptide.

As used herein, the term “oligonucleotide,” refers to a short length ofsingle-stranded polynucleotide chain. Oligonucleotides are typicallyless than 200 residues long (e.g., between 15 and 100), however, as usedherein, the term is also intended to encompass longer polynucleotidechains. Oligonucleotides are often referred to by their length. Forexample a 24 residue oligonucleotide is referred to as a “24-mer”.Oligonucleotides can form secondary and tertiary structures byself-hybridizing or by hybridizing to other polynucleotides. Suchstructures can include, but are not limited to, duplexes, hairpins,cruciforms, bends, and triplexes.

As used herein, the term “probe” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, recombinantly or by PCR amplification, which is capableof hybridizing to at least a portion of another oligonucleotide ofinterest. A probe may be single-stranded or double-stranded. Probes areuseful in the detection, identification and isolation of particular genesequences. It is contemplated that any probe used in methods of thepresent disclosure will be labeled with any “reporter molecule,” so thatis detectable in any detection system, including, but not limited toenzyme (e.g., ELISA, as well as enzyme-based histochemical assays),fluorescent, radioactive, and luminescent systems. It is not intendedthat the methods or reagents of the present disclosure be limited to anyparticular detection system or label.

The term “isolated” when used in relation to a nucleic acid, as in “anisolated oligonucleotide” or “isolated polynucleotide” refers to anucleic acid sequence that is identified and separated from at least onecomponent or contaminant with which it is ordinarily associated in itsnatural source. An isolated nucleic acid is present in a form or settingthat is different from that in which it is found in nature. In contrast,non-isolated nucleic acids are found in the state they exist in nature.For example, a given DNA sequence (e.g., a gene) is found on the hostcell chromosome in proximity to neighboring genes; RNA sequences, suchas a specific mRNA sequence encoding a specific protein, are found inthe cell as a mixture with numerous other mRNAs that encode a multitudeof proteins. However, isolated nucleic acid encoding a given proteinincludes, by way of example, such nucleic acid in cells ordinarilyexpressing the given protein where the nucleic acid is in a chromosomallocation different from that of natural cells, or is otherwise flankedby a different nucleic acid sequence than that found in nature. Theisolated nucleic acid, oligonucleotide, or polynucleotide may be presentin single-stranded or double-stranded form. When an isolated nucleicacid, oligonucleotide or polynucleotide is to be utilized to express aprotein, the nucleic acid, oligonucleotide or polynucleotide often willcontain, at a minimum, the sense or coding strand (i.e., theoligonucleotide or polynucleotide may be single-stranded), but maycontain both the sense and anti-sense strands (i.e., the oligonucleotideor polynucleotide may be double-stranded).

As used herein, the term “purified” or “to purify” refers to the removalof components (e.g., contaminants) from a sample. For example,antibodies are purified by removal of contaminating non-immunoglobulinproteins; they are also purified by the removal of immunoglobulin thatdoes not bind to the target molecule. The removal of non-immunoglobulinproteins and/or the removal of immunoglobulins that do not bind to thetarget molecule results in an increase in the percent of target-reactiveimmunoglobulins in the sample. In another example, recombinantpolypeptides are expressed in bacterial host cells and the polypeptidesare purified by the removal of host cell proteins; the percent ofrecombinant polypeptides is thereby increased in the sample.

As used herein, the term “sample” is used in its broadest sense. In onesense, it is meant to include a specimen or culture obtained from anysource, as well as biological and environmental samples. Biologicalsamples may be obtained from animals (including humans) and encompassfluids, solids, tissues, and gases. Biological samples include bloodproducts, such as plasma, serum and the like. Such examples are nothowever to be construed as limiting the sample types applicable to thepresent invention.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure is based on the discovery of recurrent genefusions in prostate cancer. The present disclosure provides diagnostic,research, and therapeutic methods that either directly or indirectlydetect or target the gene fusions. The present disclosure also providescompositions for diagnostic, research, and therapeutic purposes.

I. Gene Fusions

The present disclosure identifies recurrent gene fusions indicative ofcancer (e.g., melanoma or gastric cancer). In some embodiments, the genefusions are the result of a chromosomal rearrangement of atranscriptional regulatory region of a first gene (e.g., an androgenregulated gene or other gene) or a RAF family member gene and an RAFfamily member gene or other gene. The gene fusions typically comprise a5′ portion from a transcriptional regulatory region of first gene (e.g.,SLC45A3, RAF family member gene, AGTRAP or ESRP1) and a 3′ portion froman RAF family member gene or ESRP1gene. The recurrent gene fusions haveuse as diagnostic markers and clinical targets for cancer.

In some embodiments, the 5′ fusion partner is a transcriptional regionof an androgen regulated gene. Genes regulated by androgenic hormonesare of critical importance for the normal physiological function of thehuman prostate gland. They also contribute to the development andprogression of prostate carcinoma. Recognized ARGs include, but are notlimited to: TMPRSS2; SLC45A3; HERV-K_22q11.23; C15ORF21; FLJ35294;CANT1; PSA; PSMA; KLK2; SNRK; Seladin-1; and, FKBP51 (Paoloni-Giacobinoet al., Genomics 44: 309 (1997); Velasco et al., Endocrinology 145(8):3913 (2004)).

SLC45A3, also known as prostein or P501 S, has been shown to beexclusively expressed in normal prostate and prostate cancer at both thetranscript and protein level (Kalos et al., Prostate 60, 246-56 (2004);Xu et al., Cancer Res 61, 1563-8 (2001)).

In some embodiments, gene fusions of the present disclosure comprisetranscriptional regulatory regions of an ARG. The transcriptionalregulatory region of an ARG may contain coding or non-coding regions ofthe ARG, including the promoter region. The promoter region of the ARGmay further comprise an androgen response element) of the ARG.

In other embodiments, 5′ fusion partners comprise a portion (e.g., atranscriptional regulatory region) of an Type-1 angiotensin IIreceptor-associated protein (AGTRAP; NM_001040194, NM_001040195,NM_001040196 and NM_001040197) or epithelial splicing regulatory protein1 (ESRP1; NM_017697) or a RAF family member gene.

In some embodiments, the 3′ or 5′ fusion partner comprises at least aportion of an ESRP1 gene or a RAF family member gene.

The BRAF gene makes a protein called B-RAF, which is involved in sendingsignals in cells and in cell growth. See, e.g., Ikawa et al., Mol. CellBiol. 8(6):2651-54 (1988). This protein belongs to the raf/mil family ofserine/threonine protein kinases. Though not desiring to be bound bytheory, this protein is known to plays a role in regulating the MAPkinase/ERKs signaling pathway, which affects cell division,differentiation, and secretion. The BRAF gene may be mutated in avariety of cancer types, which causes a change in the B-RAF protein.See, e.g., Davies et al., Nature 417 (6892): 949-54 (2002); Wan et al.Cell 116:855-867 (2004) This can increase the growth and spread ofcancer cells.

Mutations in this gene have been associated with cardiofaciocutaneoussyndrome, a disease characterized by heart defects, mental retardationand a distinctive facial appearance. Mutations in this gene have alsobeen associated with various cancers, including non-Hodgkin's lymphoma,colorectal cancer, malignant melanoma, thyroid carcinoma, non-small celllung carcinoma, and adenocarcinoma of the lung.

c-raf is a gene that encodes a protein kinase called “Raf-1.”. The Raf-1protein functions in the MAPK/ERK signal transduction pathway as part ofa protein kinase cascade. Raf-1 is a serine/threonine-specific kinase.Raf-1 is a MAP kinase kinase kinase (MAP3K) which functions downstreamof the Ras family of membrane associated GTPases to which it bindsdirectly. Activated Raf-1 can phosphorylate to activate the dualspecificity protein kinases MEK1 and MEK2, which in turn phosphorylateto activate the serine/threonine specific protein kinases ERK1 and ERK2.Activated ERKs are pleiotropic effectors of cell physiology and play animportant role in the control of gene expression involved in the celldivision cycle, apoptosis, cell differentiation and cell migration.

The first identified raf gene is the oncogene v-raf (Mark et al., (Apr.1984). Science 224 (4646): 285-9). Normal (non-oncogenic) cellularhomologs of v-raf were soon found to be conserved components ofeukaryotic genomes and it was shown that they could mutate and becomeoncogenes (Shimizu et al., (1986). Int. Symp. Princess Takamatsu CancerRes. Fund 17: 85-91). A-Raf and B-Raf are two protein kinases withsimilar sequences to Raf-1. Mutations in B-Raf genes are found inseveral types of cancer (See, e.g., Davies et al., Nature 417 (6892):949-54 (2002)). The Raf kinases are targets for anticancer drugdevelopment (Sridhar et al., (Apr. 2005). Mol. Cancer Ther. 4 (4):677-85). There are several quantitative immunochemical methods availableto detect Raf kinase inhibiting drugs (Olive (October 2004). Expert RevProteomics 1 (3): 327-41).

Human BRAF DNA has the nucleotide sequence described by GenbankAccession No. NG_007873. Human BRAF mRNA has the nucleotide sequencedescribed by Genbank Accession No. NM_004333.

Human RAF1 DNA has the nucleotide sequence described by GenbankAccession No. NG_007467. Human RAF1 mRNA has the nucleotide sequencedescribed by Genbank Accession No. NM_002880.

II. Antibodies

The gene fusion proteins of the present disclosure, including fragments,derivatives and analogs thereof, may be used as immunogens to produceantibodies having use in the diagnostic, screening, research, andtherapeutic methods described below. The antibodies may be polyclonal ormonoclonal, chimeric, humanized, single chain, Fv or Fab fragments.Various procedures known to those of ordinary skill in the art may beused for the production and labeling of such antibodies and fragments.See, e.g., Burns, ed., Immunochemical Protocols, 3^(rd) ed., HumanaPress (2005); Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory (1988); Kozbor et al., Immunology Today 4: 72(1983); Köhler and Milstein, Nature 256: 495 (1975). Antibodies orfragments exploiting the differences between the truncated or chimericprotein resulting from a gene fusion and their respective nativeproteins are particularly preferred.

III. Diagnostic and Screening Applications

The gene fusions described herein may be detectable as DNA, RNA orprotein. Initially, the gene fusion is detectable as a chromosomalrearrangement of genomic DNA having a 5′ portion from a first gene and a3′ portion from a second gene. Once transcribed, the gene fusion isdetectable as a chimeric mRNA having a 5′ portion from a first gene anda 3′ portion from a second gene. Once translated, the gene fusion isdetectable as fusion of a 5′ portion from a first protein and a 3′portion from a second protein or a truncated version of a first orsecond protein. The truncated or fusion proteins may differ from theirrespective native proteins in amino acid sequence, post-translationalprocessing and/or secondary, tertiary or quaternary structure. Suchdifferences, if present, can be used to identify the presence of thegene fusion. Specific methods of detection are described in more detailbelow.

The present disclosure provides DNA, RNA and protein based diagnosticand screening methods that either directly or indirectly detect the genefusions. The present disclosure also provides compositions and kits fordiagnostic and screening purposes.

The diagnostic and screening methods of the present disclosure may bequalitative or quantitative. Quantitative methods may be used, forexample, to discriminate between indolent and aggressive cancers via acutoff or threshold level. Where applicable, qualitative or quantitativemethods of embodiments of the disclosure include amplification of atarget, a signal or an intermediary (e.g., a universal primer).

An initial assay may confirm the presence of a gene fusion but notidentify the specific fusion. A secondary assay may then be performed todetermine the identity of the particular fusion, if desired. The secondassay may use a different detection technology than the initial assay.

The gene fusions may be detected along with other markers in a multiplexor panel format. Markers are selected for their predictive value aloneor in combination with the gene fusions. Exemplary prostate cancermarkers include, but are not limited to: AMACR/P504S (U.S. Pat. No.6,262,245); PCA3 (U.S. Pat. No. 7,008,765); PCGEM1 (U.S. Pat. No.6,828,429); prostein/P501S, P503S, P504S, P509S, P510S, prostase/P703P,P710P (U.S. Publication No. 20030185830); RAS/KRAS (Bos, Cancer Res.49:4682-89 (1989); Kranenburg, Biochimica et Biophysica Acta 1756:81-82(2005)); and, those disclosed in U.S. Pat. Nos. 5,854,206 and 6,034,218,7,229,774, each of which is herein incorporated by reference in itsentirety. Markers for other cancers, diseases, infections, and metabolicconditions are also contemplated for inclusion in a multiplex or panelformat.

The diagnostic methods of the present disclosure may also be modifiedwith reference to data correlating particular gene fusions with thestage, aggressiveness or progression of the disease or the presence orrisk of metastasis. Ultimately, the information provided will assist aphysician in choosing the best course of treatment for a particularpatient.

A. Sample

Any sample suspected of containing the gene fusions may be testedaccording to the methods of the present disclosure. By way ofnon-limiting example, the sample may be tissue (e.g., a prostate biopsysample or a tissue sample obtained by prostatectomy, a gastric biopsysample, or a skin sample), blood, urine, semen, cells, fecal amples,cell secretions or a fraction thereof (e.g., plasma, serum, exosomes,urine supernatant, or urine cell pellet). A urine sample is preferablycollected immediately following an attentive digital rectal examination(DRE), which causes prostate cells from the prostate gland to shed intothe urinary tract.

In some embodiments, skin and/or gastic samples are obtained using knownmethods. For example, skin samples are generally obtained via biopsy ofskin cells. There are four main types of skin biopsies: shave biopsy,punch biopsy, excisional biopsy, and incisional biopsy. All involvescraping or cutting a small sample of skin for analysis. Gastric cellsamples are generally obtained via endoscopic or surgical biopsy or viaa fecal sample.

The patient sample typically involves preliminary processing designed toisolate or enrich the sample for the gene fusion(s) or cells thatcontain the gene fusion(s). A variety of techniques known to those ofordinary skill in the art may be used for this purpose, including butnot limited to: centrifugation; immunocapture; cell lysis; and, nucleicacid target capture (See, e.g., EP Pat. No. 1 409 727, hereinincorporated by reference in its entirety).

B. DNA and RNA Detection

The gene fusions of the present disclosure may be detected aschromosomal rearrangements of genomic DNA or chimeric mRNA using avariety of nucleic acid techniques known to those of ordinary skill inthe art, including but not limited to: nucleic acid sequencing; nucleicacid hybridization; and, nucleic acid amplification.

1. Sequencing

Illustrative non-limiting examples of nucleic acid sequencing techniquesinclude, but are not limited to, chain terminator (Sanger) sequencingand dye terminator sequencing, or high throughput sequencing methods.The present disclosure is not intended to be limited to any particularmethods of sequencing. Those of ordinary skill in the art will recognizethat because RNA is less stable in the cell and more prone to nucleaseattack experimentally RNA is usually reverse transcribed to DNA beforesequencing.

Chain terminator sequencing uses sequence-specific termination of a DNAsynthesis reaction using modified nucleotide substrates. Extension isinitiated at a specific site on the template DNA by using a shortradioactive, or other labeled, oligonucleotide primer complementary tothe template at that region. The oligonucleotide primer is extendedusing a DNA polymerase, standard four deoxynucleotide bases, and a lowconcentration of one chain terminating nucleotide, most commonly adi-deoxynucleotide. This reaction is repeated in four separate tubeswith each of the bases taking turns as the di-deoxynucleotide. Limitedincorporation of the chain terminating nucleotide by the DNA polymeraseresults in a series of related DNA fragments that are terminated only atpositions where that particular di-deoxynucleotide is used. For eachreaction tube, the fragments are size-separated by electrophoresis in aslab polyacrylamide gel or a capillary tube filled with a viscouspolymer. The sequence is determined by reading which lane produces avisualized mark from the labeled primer as you scan from the top of thegel to the bottom.

Dye terminator sequencing alternatively labels the terminators. Completesequencing can be performed in a single reaction by labeling each of thedi-deoxynucleotide chain-terminators with a separate fluorescent dye,which fluoresces at a different wavelength.

A variety of nucleic acid sequencing methods are contemplated for use inthe methods of the present disclosure including, for example, chainterminator (Sanger) sequencing, dye terminator sequencing, andhigh-throughput sequencing methods. Many of these sequencing methods arewell known in the art. See, e.g., Sanger et al., Proc. Natl. Acad. Sci.USA 74:5463-5467 (1997); Maxam et al., Proc. Natl. Acad. Sci. USA74:560-564 (1977); Drmanac, et al., Nat. Biotechnol. 16:54-58 (1998);Kato, Int. J. Clin. Exp. Med. 2:193-202 (2009); Ronaghi et al., Anal.Biochem. 242:84-89 (1996); Margulies et al., Nature 437:376-380 (2005);Ruparel et al., Proc. Natl. Acad. Sci. USA 102:5932-5937 (2005), andHarris et al., Science 320:106-109 (2008); Levene et al., Science299:682-686 (2003); Korlach et al., Proc. Natl. Acad. Sci. USA105:1176-1181 (2008); Branton et al., Nat. Biotechnol. 26(10):1146-53(2008); Eid et al., Science 323:133-138 (2009); each of which is hereinincorporated by reference in its entirety.

2. Hybridization

Illustrative non-limiting examples of nucleic acid hybridizationtechniques include, but are not limited to, in situ hybridization (ISH),microarray, and Southern or Northern blot.

In situ hybridization (ISH) is a type of hybridization that uses alabeled complementary DNA or RNA strand as a probe to localize aspecific DNA or RNA sequence in a portion or section of tissue (insitu), or, if the tissue is small enough, the entire tissue (whole mountISH). DNA ISH can be used to determine the structure of chromosomes. RNAISH is used to measure and localize mRNAs and other transcripts withintissue sections or whole mounts. Sample cells and tissues are usuallytreated to fix the target transcripts in place and to increase access ofthe probe. The probe hybridizes to the target sequence at elevatedtemperature, and then the excess probe is washed away. The probe thatwas labeled with radio-, fluorescent- or antigen-labeled bases islocalized and quantitated in the tissue using autoradiography,fluorescence microscopy or immunohistochemistry. ISH can also use two ormore probes, labeled with radioactivity or the other non-radioactivelabels, to simultaneously detect two or more transcripts.

a. FISH

In some embodiments, fusion sequences are detected using fluorescence insitu hybridization (FISH). The preferred FISH assays for methods ofembodiments of the present disclosure utilize bacterial artificialchromosomes (BACs). These have been used extensively in the human genomesequencing project (see Nature 409: 953-958 (2001)) and clonescontaining specific BACs are available through distributors that can belocated through many sources, e.g., NCBI. Each BAC clone from the humangenome has been given a reference name that unambiguously identifies it.These names can be used to find a corresponding GenBank sequence and toorder copies of the clone from a distributor.

b. Microarrays

Different kinds of biological assays are called microarrays including,but not limited to: DNA microarrays (e.g., cDNA microarrays andoligonucleotide microarrays); protein microarrays; tissue microarrays;transfection or cell microarrays; chemical compound microarrays; and,antibody microarrays. A DNA microarray, commonly known as gene chip, DNAchip, or biochip, is a collection of microscopic DNA spots attached to asolid surface (e.g., glass, plastic or silicon chip) forming an arrayfor the purpose of expression profiling or monitoring expression levelsfor thousands of genes simultaneously. The affixed DNA segments areknown as probes, thousands of which can be used in a single DNAmicroarray. Microarrays can be used to identify disease genes bycomparing gene expression in disease and normal cells. Microarrays canbe fabricated using a variety of technologies, including but not limitedto: printing with fine-pointed pins onto glass slides; photolithographyusing pre-made masks; photolithography using dynamic micromirrordevices; ink-jet printing; or, electrochemistry on microelectrodearrays.

Southern and Northern blotting may be used to detect specific DNA or RNAsequences, respectively. In these techniques DNA or RNA is extractedfrom a sample, fragmented, electrophoretically separated on a matrixgel, and transferred to a membrane filter. The filter bound DNA or RNAis subject to hybridization with a labeled probe complementary to thesequence of interest. Hybridized probe bound to the filter is detected.A variant of the procedure is the reverse Northern blot, in which thesubstrate nucleic acid that is affixed to the membrane is a collectionof isolated DNA fragments and the probe is RNA extracted from a tissueand labeled.

3. Amplification

Chromosomal rearrangements of genomic DNA and chimeric mRNA may beamplified prior to or simultaneous with detection. Illustrativenon-limiting examples of nucleic acid amplification techniques include,but are not limited to, polymerase chain reaction (PCR), reversetranscription polymerase chain reaction (RT-PCR), transcription-mediatedamplification (TMA), ligase chain reaction (LCR), strand displacementamplification (SDA), and nucleic acid sequence based amplification(NASBA). Those of ordinary skill in the art will recognize that certainamplification techniques (e.g., PCR) require that RNA be reversedtranscribed to DNA prior to amplification (e.g., RT-PCR), whereas otheramplification techniques directly amplify RNA (e.g., TMA and NASBA).

The polymerase chain reaction (U.S. Pat. Nos. 4,683,195, 4,683,202,4,800,159 and 4,965,188, each of which is herein incorporated byreference in its entirety), commonly referred to as PCR, uses multiplecycles of denaturation, annealing of primer pairs to opposite strands,and primer extension to exponentially increase copy numbers of a targetnucleic acid sequence. In a variation called RT-PCR, reversetranscriptase (RT) is used to make a complementary DNA (cDNA) from mRNA,and the cDNA is then amplified by PCR to produce multiple copies of DNA.For other various permutations of PCR see, e.g., U.S. Pat. Nos.4,683,195, 4,683,202 and 4,800,159; Mullis et al., Meth. Enzymol. 155:335 (1987); and, Murakawa et al., DNA 7: 287 (1988), each of which isherein incorporated by reference in its entirety.

Transcription mediated amplification (U.S. Pat. Nos. 5,480,784 and5,399,491, each of which is herein incorporated by reference in itsentirety), commonly referred to as TMA, synthesizes multiple copies of atarget nucleic acid sequence autocatalytically under conditions ofsubstantially constant temperature, ionic strength, and pH in whichmultiple RNA copies of the target sequence autocatalytically generateadditional copies. See, e.g., U.S. Pat. Nos. 5,399,491 and 5,824,518,each of which is herein incorporated by reference in its entirety. In avariation described in U.S. Pat. No. 7,374,885 (herein incorporated byreference in its entirety), TMA optionally incorporates the use ofblocking moieties, terminating moieties, and other modifying moieties toimprove TMA process sensitivity and accuracy.

The ligase chain reaction (Weiss, R., Science 254: 1292 (1991), hereinincorporated by reference in its entirety), commonly referred to as LCR,uses two sets of complementary DNA oligonucleotides that hybridize toadjacent regions of the target nucleic acid. The DNA oligonucleotidesare covalently linked by a DNA ligase in repeated cycles of thermaldenaturation, hybridization and ligation to produce a detectabledouble-stranded ligated oligonucleotide product.

Strand displacement amplification (Walker, G. et al., Proc. Natl. Acad.Sci. USA 89: 392-396 (1992); U.S. Pat. Nos. 5,270,184 and 5,455,166,each of which is herein incorporated by reference in its entirety),commonly referred to as SDA, uses cycles of annealing pairs of primersequences to opposite strands of a target sequence, primer extension inthe presence of a dNTPaS to produce a duplex hemiphosphorothioatedprimer extension product, endonuclease-mediated nicking of ahemimodified restriction endonuclease recognition site, andpolymerase-mediated primer extension from the 3′ end of the nick todisplace an existing strand and produce a strand for the next round ofprimer annealing, nicking and strand displacement, resulting ingeometric amplification of product. Thermophilic SDA (tSDA) usesthermophilic endonucleases and polymerases at higher temperatures inessentially the same method (EP Pat. No. 0 684 315).

Other amplification methods include, for example: nucleic acid sequencebased amplification (U.S. Pat. No. 5,130,238, herein incorporated byreference in its entirety), commonly referred to as NASBA; one that usesan RNA replicase to amplify the probe molecule itself (Lizardi et al.,BioTechnol. 6: 1197 (1988), herein incorporated by reference in itsentirety), commonly referred to as Qβ replicase; a transcription basedamplification method (Kwoh et al., Proc. Natl. Acad. Sci. USA 86:1173(1989)); and, self-sustained sequence replication (Guatelli et al.,Proc. Natl. Acad. Sci. USA 87: 1874 (1990), each of which is hereinincorporated by reference in its entirety). For further discussion ofknown amplification methods see Persing, David H., “In Vitro NucleicAcid Amplification Techniques” in Diagnostic Medical Microbiology:Principles and Applications (Persing et al., Eds.), pp. 51-87 (AmericanSociety for Microbiology, Washington, D.C. (1993)).

4. Detection Methods

Non-amplified or amplified gene fusion nucleic acids can be detected byany conventional means. For example, the gene fusions can be detected byhybridization with a detectably labeled probe and measurement of theresulting hybrids. Illustrative non-limiting examples of detectionmethods are described below.

One illustrative detection method, the Hybridization Protection Assay(HPA) involves hybridizing a chemiluminescent oligonucleotide probe(e.g., an acridinium ester-labeled (AE) probe) to the target sequence,selectively hydrolyzing the chemiluminescent label present onunhybridized probe, and measuring the chemiluminescence produced fromthe remaining probe in a luminometer. See, e.g., U.S. Pat. No.5,283,174; Nelson et al., Nonisotopic Probing, Blotting, and Sequencing,ch. 17 (Larry J. Kricka ed., 2d ed. 1995, each of which is hereinincorporated by reference in its entirety).

Another illustrative detection method provides for quantitativeevaluation of the amplification process in real-time. Evaluation of anamplification process in “real-time” involves determining the amount ofamplicon in the reaction mixture either continuously or periodicallyduring the amplification reaction, and using the determined values tocalculate the amount of target sequence initially present in the sample.A variety of methods for determining the amount of initial targetsequence present in a sample based on real-time amplification are wellknown in the art. These include methods disclosed in U.S. Pat. Nos.6,303,305 and 6,541,205, each of which is herein incorporated byreference in its entirety. Another method for determining the quantityof target sequence initially present in a sample, but which is not basedon a real-time amplification, is disclosed in U.S. Pat. No. 5,710,029,herein incorporated by reference in its entirety.

Amplification products may be detected in real-time through the use ofvarious self-hybridizing probes, most of which have a stem-loopstructure. Such self-hybridizing probes are labeled so that they emitdifferently detectable signals, depending on whether the probes are in aself-hybridized state or an altered state through hybridization to atarget sequence. By way of non-limiting example, “molecular torches” area type of self-hybridizing probe that includes distinct regions ofself-complementarity (referred to as “the target binding domain” and“the target closing domain”) which are connected by a joining region(e.g., non-nucleotide linker) and which hybridize to each other underpredetermined hybridization assay conditions. In a preferred embodiment,molecular torches contain single-stranded base regions in the targetbinding domain that are from 1 to about 20 bases in length and areaccessible for hybridization to a target sequence present in anamplification reaction under strand displacement conditions. Understrand displacement conditions, hybridization of the two complementaryregions, which may be fully or partially complementary, of the moleculartorch is favored, except in the presence of the target sequence, whichwill bind to the single-stranded region present in the target bindingdomain and displace all or a portion of the target closing domain. Thetarget binding domain and the target closing domain of a molecular torchinclude a detectable label or a pair of interacting labels (e.g.,luminescent/quencher) positioned so that a different signal is producedwhen the molecular torch is self-hybridized than when the moleculartorch is hybridized to the target sequence, thereby permitting detectionof probe:target duplexes in a test sample in the presence ofunhybridized molecular torches. Molecular torches and a variety of typesof interacting label pairs, including fluorescence resonance energytransfer (FRET) labels, are disclosed in, for example U.S. Pat. Nos.6,534,274 and 5,776,782, each of which is herein incorporated byreference in its entirety.

The interaction between two molecules can also be detected, e.g., usingfluorescence energy transfer (FRET) (see, for example, Lakowicz et al.,U.S. Pat. No. 5,631,169; Stavrianopoulos et al., U.S. Pat. No.4,968,103; each of which is herein incorporated by reference). Afluorophore label is selected such that a first donor molecule's emittedfluorescent energy will be absorbed by a fluorescent label on a second,‘acceptor’ molecule, which in turn is able to fluoresce due to theabsorbed energy.

Alternately, the ‘donor’ protein molecule may simply utilize the naturalfluorescent energy of tryptophan residues. Labels are chosen that emitdifferent wavelengths of light, such that the ‘acceptor’ molecule labelmay be differentiated from that of the ‘donor’. Since the efficiency ofenergy transfer between the labels is related to the distance separatingthe molecules, the spatial relationship between the molecules can beassessed. In a situation in which binding occurs between the molecules,the fluorescent emission of the ‘acceptor’ molecule label should bemaximal. A FRET binding event can be conveniently measured throughstandard fluorometric detection means well known in the art (e.g., usinga fluorimeter).

Another example of a detection probe having self-complementarity is a“molecular beacon.” Molecular beacons include nucleic acid moleculeshaving a target complementary sequence, an affinity pair (or nucleicacid arms) holding the probe in a closed conformation in the absence ofa target sequence present in an amplification reaction, and a label pairthat interacts when the probe is in a closed conformation. Hybridizationof the target sequence and the target complementary sequence separatesthe members of the affinity pair, thereby shifting the probe to an openconformation. The shift to the open conformation is detectable due toreduced interaction of the label pair, which may be, for example, afluorophore and a quencher (e.g., DABCYL and EDANS). Molecular beaconsare disclosed, for example, in U.S. Pat. Nos. 5,925,517 and 6,150,097,herein incorporated by reference in its entirety.

Other self-hybridizing probes are well known to those of ordinary skillin the art. By way of non-limiting example, probe binding pairs havinginteracting labels, such as those disclosed in U.S. Pat. No. 5,928,862(herein incorporated by reference in its entirety) might be adapted foruse in method of embodiments of the present disclosure. Probe systemsused to detect single nucleotide polymorphisms (SNPs) might also beutilized in the present invention. Additional detection systems include“molecular switches,” as disclosed in U.S. Publ. No. 20050042638, hereinincorporated by reference in its entirety. Other probes, such as thosecomprising intercalating dyes and/or fluorochromes, are also useful fordetection of amplification products methods of embodiments of thepresent disclosure. See, e.g., U.S. Pat. No. 5,814,447 (hereinincorporated by reference in its entirety).

C. Protein Detection

The gene fusions of the present disclosure may be detected as truncatedor chimeric proteins using a variety of protein techniques known tothose of ordinary skill in the art, including but not limited to:protein sequencing and immunoassays.

1. Sequencing

Illustrative non-limiting examples of protein sequencing techniquesinclude, but are not limited to, mass spectrometry and Edmandegradation.

Mass spectrometry can, in principle, sequence any size protein. Aprotein is digested by an endoprotease, and the resulting solution ispassed through a high pressure liquid chromatography column. At the endof this column, the solution is sprayed out of a narrow nozzle chargedto a high positive potential into the mass spectrometer. The charge onthe droplets causes them to fragment until only single ions remain. Thepeptides are then fragmented and the mass-charge ratios of the fragmentsmeasured. The mass spectrum is analyzed by computer and often comparedagainst a database of previously sequenced proteins in order todetermine the sequences of the fragments. The process is then repeatedwith a different digestion enzyme, and the overlaps in sequences areused to construct a sequence for the protein.

In the Edman degradation reaction (see, e.g., Edman, Acta Chem. Scand.4:283-93 (1950)), the peptide to be sequenced is adsorbed onto a solidsurface (e.g., a glass fiber coated with polybrene). Though there arevarious well known modifications to this procedure (including automatedmodifications), one exemplary method involves the use of the Edmanreagent, phenylisothiocyanate (PITC), which is added, together with amildly basic buffer solution of 12% trimethylamine, to an adsorbedpeptide, and which reacts with the amine group of the N-terminal aminoacid of the adsorbed peptide. The terminal amino acid derivative canthen be selectively detached by the addition of anhydrous acid. Thederivative isomerizes to give a substituted phenylthiohydantoin, whichcan be washed off and identified by chromatography, and the cycle can berepeated. The efficiency of each step is about or over 98%, which allowsabout 50 amino acids to be reliably determined.

2. Immunoassays Illustrative non-limiting examples of immunoassaysinclude, but are not limited to: immunoprecipitation; Western blot;ELISA; immunohistochemistry; immunocytochemistry; immunochromatography;flow cytometry; and, immuno-PCR. Polyclonal or monoclonal antibodiesdetectably labeled using various techniques known to those of ordinaryskill in the art (e.g., colorimetric, fluorescent, chemiluminescent orradioactive labels) are suitable for use in the immunoassays.

Immunoprecipitation is the technique of precipitating an antigen out ofsolution using an antibody specific to that antigen. The process can beused to identify proteins or protein complexes present in cell extractsby targeting a specific protein or a protein believed to be in thecomplex. The complexes are brought out of solution by insolubleantibody-binding proteins isolated initially from bacteria, such asProtein A and Protein G. The antibodies can also be coupled to sepharosebeads that can easily be isolated out of solution. After washing, theprecipitate can be analyzed using mass spectrometry, Western blotting,or any number of other methods for identifying constituents in thecomplex.

A Western blot, or immunoblot, is a method to detect protein in a givensample of tissue homogenate or extract. It uses gel electrophoresis toseparate denatured proteins by mass. The proteins are then transferredout of the gel and onto a membrane, typically polyvinyldiflroride ornitrocellulose, where they are probed using antibodies specific to theprotein of interest. As a result, researchers can examine the amount ofprotein in a given sample and compare levels between several groups.

An ELISA, short for Enzyme-Linked ImmunoSorbent Assay, is a biochemicaltechnique to detect the presence of an antibody or an antigen in asample. It utilizes a minimum of two antibodies, one of which isspecific to the antigen and the other of which is coupled to an enzyme.

The second antibody will cause a chromogenic or fluorogenic substrate toproduce a signal. Variations of ELISA include sandwich ELISA,competitive ELISA, and ELISPOT. Because the ELISA can be performed toevaluate either the presence of antigen or the presence of antibody in asample, it is a useful tool both for determining serum antibodyconcentrations and also for detecting the presence of antigen.

Immunohistochemistry and immunocytochemistry refer to the process oflocalizing proteins in a tissue section or cell, respectively, via theprinciple of antigens in tissue or cells binding to their respectiveantibodies. Visualization is enabled by tagging the antibody with colorproducing or fluorescent tags. Typical examples of color tags include,but are not limited to, horseradish peroxidase and alkaline phosphatase.Typical examples of fluorophore tags include, but are not limited to,fluorescein isothiocyanate (FITC) or phycoerythrin (PE).

Flow cytometry is a technique for counting, examining and optionallysorting microscopic particles or cells suspended in a stream of fluid.It allows simultaneous multiparametric analysis of the physical and/orchemical characteristics of single cells flowing through anoptical/electronic detection apparatus. A beam of light (e.g., a laser)of a single frequency or color is directed onto a hydrodynamicallyfocused stream of fluid. A number of detectors are aimed at the pointwhere the stream passes through the light beam; one in line with thelight beam (Forward Scatter or FSC) and several perpendicular to it(Side Scatter (SSC) and one or more fluorescent detectors). Eachsuspended particle passing through the beam scatters the light in someway, and fluorescent chemicals in the particle may be excited intoemitting light at a lower frequency than the light source. Thecombination of scattered and fluorescent light is picked up by thedetectors, and by analyzing fluctuations in brightness at each detector,one for each fluorescent emission peak, it is possible to deduce variousfacts about the physical and chemical structure of each individualparticle. FSC correlates with the cell volume and SSC correlates withthe density or inner complexity of the particle (e.g., shape of thenucleus, the amount and type of cytoplasmic granules or the membraneroughness).

Immuno-polymerase chain reaction (IPCR) utilizes nucleic acidamplification techniques to increase signal generation in antibody-basedimmunoassays. Because no protein equivalence of PCR exists, that is,proteins cannot be replicated in the same manner that nucleic acid isreplicated during PCR, the only way to increase detection sensitivity isby signal amplification. The target proteins are bound to antibodieswhich are directly or indirectly conjugated to oligonucleotides. Unboundantibodies are washed away and the remaining bound antibodies have theiroligonucleotides amplified. Protein detection occurs via detection ofamplified oligonucleotides using standard nucleic acid detectionmethods, including real-time methods.

D. Data Analysis

In some embodiments, a computer-based analysis program is used totranslate the raw data generated by the detection assay (e.g., thepresence, absence, or amount of a given gene fusion or other markers)into data of predictive value for a clinician. The clinician can accessthe predictive data using any suitable means. Thus, in some preferredembodiments, the present disclosure provides the further benefit thatthe clinician, who may not be specifically trained in genetics ormolecular biology, need not understand the raw data. The data is can bepresented directly to the clinician in its most useful form. Theclinician is may then be then able to immediately utilize theinformation in order to optimize the care of the subject.

The present disclosure contemplates any method capable of receiving,processing, and transmitting the information to and from laboratoriesconducting the assays, medical personal, and subjects. For example, insome embodiments of the present invention, a sample (e.g., a biopsy or aserum or urine sample) is obtained from a subject and submitted to aprofiling service (e.g., clinical lab at a medical facility, genomicprofiling business, etc.), located in any part of the world (e.g., in acountry different than the country where the subject resides or wherethe information is ultimately used) to generate raw data. Where thesample comprises a tissue or other biological sample, the subject mayvisit a medical center to have the sample obtained and sent to theprofiling center, or subjects may collect the sample themselves (e.g., aurine sample) and directly send it to a profiling center. Where thesample comprises previously determined biological information, theinformation may be directly sent to the profiling service by the subject(e.g., an information card containing the information may be scanned bya computer and the data transmitted to a computer of the profilingcenter using an electronic communication systems). Once received by theprofiling service, the sample is processed and a profile is produced(i.e., expression data), specific for the diagnostic or prognosticinformation desired for the subject.

The profile data may then be prepared in a format suitable forinterpretation by a treating clinician. For example, rather thanproviding raw expression data, the prepared format may represent adiagnosis or risk assessment (e.g., likelihood of cancer being present)for the subject, along with recommendations for particular treatmentoptions. The data may be displayed to the clinician by any suitablemethod. For example, in some embodiments, the profiling servicegenerates a report that can be printed for the clinician (e.g., at thepoint of care) or displayed to the clinician on a computer monitor.

In some embodiments, the information is first analyzed at the point ofcare or at a regional facility. The raw data is then sent to a centralprocessing facility for further analysis and/or to convert the raw datato information useful for a clinician or patient. The central processingfacility provides the advantage of privacy (all data is stored in acentral facility with uniform security protocols), speed, and uniformityof data analysis. The central processing facility can then control thefate of the data following treatment of the subject. For example, usingan electronic communication system, the central facility can providedata to the clinician, the subject, or researchers.

In some embodiments, the subject is able to directly access the datausing the electronic communication system. The subject may chose, forexample, further or altered intervention or counseling based on theresults. In some embodiments, the data is used for research use. Forexample, the data may be used to further optimize the inclusion orelimination of markers as useful indicators of a particular condition orstage of disease.

E. In Vivo Imaging

The gene fusions of the present disclosure may also be detected using invivo imaging techniques, including but not limited to: radionuclideimaging; positron emission tomography (PET); computerized axialtomography, X-ray or magnetic resonance imaging methods, fluorescencedetection, and chemiluminescent detection. In some embodiments, in vivoimaging techniques are used to visualize the presence of or expressionof cancer markers in an animal (e.g., a human or non-human mammal). Forexample, in some embodiments, cancer marker mRNA or protein is labeledusing a labeled antibody specific for the cancer marker. A specificallybound and labeled antibody can be detected in an individual using an invivo imaging method, including, but not limited to, radionuclideimaging, positron emission tomography, computerized axial tomography,X-ray or magnetic resonance imaging method, fluorescence detection, andchemiluminescent detection. Methods for generating antibodies to thecancer markers of the present disclosure are described below.

The in vivo imaging methods of the present disclosure are useful in thediagnosis of cancers that express the cancer markers of the presentinvention (e.g., prostate cancer, gastric cancer, melanoma). In vivoimaging is used to visualize the presence of a marker indicative of thecancer. Such techniques allow for diagnosis without the use of anunpleasant biopsy. The in vivo imaging methods of the present disclosureare also useful for providing prognoses to cancer patients. For example,the presence of a marker indicative of cancers likely to metastasize canbe detected. The in vivo imaging methods of the present disclosure canfurther be used to detect metastatic cancers in other parts of the body.

In some embodiments, reagents (e.g., antibodies) specific for the genefusions of the present disclosure are fluorescently labeled. The labeledantibodies are introduced into a subject (e.g., orally or parenterally).Fluorescently labeled antibodies are detected using any suitable method(e.g., using the apparatus described in U.S. Pat. No. 6,198,107, hereinincorporated by reference).

In other embodiments, antibodies are radioactively labeled. The use ofantibodies for in vivo diagnosis is well known in the art. Sumerdon etal., (Nucl. Med. Biol 17:247-254 [1990] have described an optimizedantibody-chelator for the radioimmunoscintographic imaging of tumorsusing Indium-111 as the label. Griffin et al., (J Clin Onc 9:631-640[1991]) have described the use of this agent in detecting tumors inpatients suspected of having recurrent colorectal cancer. The use ofsimilar agents with paramagnetic ions as labels for magnetic resonanceimaging is known in the art (Lauffer, Magnetic Resonance in Medicine22:339-342 [1991]). The label used will depend on the imaging modalitychosen. Radioactive labels such as Indium-111, Technetium-99m, orIodine-131 can be used for planar scans or single photon emissioncomputed tomography (SPECT). Positron emitting labels such asFluorine-19 can also be used for positron emission tomography (PET). ForMRI, paramagnetic ions such as Gadolinium (III) or Manganese (II) can beused.

Radioactive metals with half-lives ranging from 1 hour to 3.5 days areavailable for conjugation to antibodies, such as scandium-47 (3.5 days)gallium-67 (2.8 days), gallium-68 (68 minutes), technetiium-99m (6hours), and indium-111 (3.2 days), of which gallium-67, technetium-99m,and indium-111 are preferable for gamma camera imaging, gallium-68 ispreferable for positron emission tomography.

A useful method of labeling antibodies with such radiometals is by meansof a bifunctional chelating agent, such as diethylenetriaminepentaaceticacid (DTPA), as described, for example, by Khaw et al. (Science 209:295[1980]) for In-111 and Tc-99m, and by Scheinberg et al. (Science215:1511 [1982]). Other chelating agents may also be used, but the1-(p-carboxymethoxybenzyl)EDTA and the carboxycarbonic anhydride of DTPAare advantageous because their use permits conjugation without affectingthe antibody's immunoreactivity substantially.

Another method for coupling DPTA to proteins is by use of the cyclicanhydride of DTPA, as described by Hnatowich et al. (Int. J. Appl.Radiat. Isot. 33:327 [1982]) for labeling of albumin with In-111, butwhich can be adapted for labeling of antibodies. A suitable method oflabeling antibodies with Tc-99m which does not use chelation with DPTAis the pretinning method of Crockford et al., (U.S. Pat. No. 4,323,546,herein incorporated by reference).

A preferred method of labeling immunoglobulins with Tc-99m is thatdescribed by Wong et al. (Int. J. Appl. Radiat. Isot., 29:251 [1978])for plasma protein, and recently applied successfully by Wong et al. (J.Nucl. Med., 23:229 [1981]) for labeling antibodies.

In the case of the radiometals conjugated to the specific antibody, itis likewise desirable to introduce as high a proportion of theradiolabel as possible into the antibody molecule without destroying itsimmunospecificity. A further improvement may be achieved by effectingradiolabeling in the presence of the specific cancer marker of thepresent invention, to insure that the antigen binding site on theantibody will be protected. The antigen is separated after labeling.

In still further embodiments, in vivo biophotonic imaging (Xenogen,Almeda, Calif.) is utilized for in vivo imaging. This real-time in vivoimaging utilizes luciferase. The luciferase gene is incorporated intocells, microorganisms, and animals (e.g., as a fusion protein with agene fusion of the present disclosure). When active, it leads to areaction that emits light. A CCD camera and software is used to capturethe image and analyze it.

F. Compositions & Kits

Any of these compositions, alone or in combination with othercompositions of the present disclosure, may be provided in the form of akit. For example, the single labeled probe and pair of amplificationoligonucleotides may be provided in a kit for the amplification anddetection of gene fusions of the present invention. Kits may furthercomprise appropriate controls and/or detection reagents. The probe andantibody compositions of the present disclosure may also be provided inthe form of an array.

Compositions for use in the diagnostic methods of the present inventioninclude, but are not limited to, probes, amplification oligonucleotides,and antibodies. Particularly preferred compositions detect a productonly when a first gene fuses to a second gene gene. These compositionsinclude: a single labeled probe comprising a sequence that hybridizes tothe junction at which a 5′ portion from a first gene fuses to a 3′portion from a second gene (i.e., spans the gene fusion junction); apair of amplification oligonucleotides wherein the first amplificationoligonucleotide comprises a sequence that hybridizes to atranscriptional regulatory region of a 5′ portion from a first genefuses to a 3′ portion from a second gene; an antibody to anamino-terminally truncated protein resulting from a fusion of a firstprotein to a second gene; or, an antibody to a chimeric protein havingan amino-terminal portion from a first gene and a carboxy-terminalportion from a second gene. Other useful compositions, however, include:a pair of labeled probes wherein the first labeled probe comprises asequence that hybridizes to a transcriptional regulatory region of afirst gene and the second labeled probe comprises a sequence thathybridizes to a second gene.

IV. Companion Diagnostics

In some embodiments, the present disclosure provides compositions andmethods for determining a treatment course of action in response to asubject's gene fusion status. For example, screening for RAF kinasefusions is useful in identifying people with cancer who benefit fromtreatment with RAF kinase inhibitors. Individuals found to a have a genefusions that comprises a RAF family member gene fusion are then treatedwith a RAF inhibitor.

The present disclosure is not limited to a particular RAF inhibitor orRAF pathway inhibitor. RAF kinase inhibitors are known in the art. Insome embodiments, inhibitors are antisense oligonucleotides, siRNA,antibodies and small molecules. Exemplary small molecule inhibitorsinclude, but are not limited to, RAF265, XL281, AZD6244, PLX4032,PLX4720, GDC 0879, AZ 628, Sorafenib (BAY43-9006) and those described inUS Pat. Pub. No. 2010/0063088 and U.S. Pat. No. 7,199,137, each of whichis herein incorporated by reference in its entirety.

BAY43-9006 has the chemical nameN-(3-trifluoromethyl-4-chlorophenyl)-N′-(4-(2-methylcarbamoylpyridin-4-yl)oxyphenyl)urea and the structure:

PLX4720 has the structure:

AZ 628 has the structure:

GDC 0879 has the structure:

PLX4032 has the structure:

RAF265 has the structure:

AZD6244 has the structure:

V. Drug Screening Applications

In some embodiments, the present disclosure provides drug screeningassays (e.g., to screen for anticancer drugs). The screening methods ofthe present disclosure utilize cancer markers identified using themethods of the present invention (e.g., including but not limited to,gene fusions of the present invention). For example, in someembodiments, the present disclosure provides methods of screening forcompounds that alter (e.g., decrease) the expression of gene fusions.The compounds or agents may interfere with transcription, byinteracting, for example, with the promoter region. The compounds oragents may interfere with mRNA produced from the fusion (e.g., by RNAinterference, antisense technologies, etc.). The compounds or agents mayinterfere with pathways that are upstream or downstream of thebiological activity of the fusion. In some embodiments, candidatecompounds are antisense or interfering RNA agents (e.g.,oligonucleotides) directed against cancer markers. In other embodiments,candidate compounds are antibodies or small molecules that specificallybind to a cancer marker regulator or expression products of the presentdisclosure and inhibit its biological function.

In one screening method, candidate compounds are evaluated for theirability to alter cancer marker expression by contacting a compound witha cell expressing a cancer marker and then assaying for the effect ofthe candidate compounds on expression. In some embodiments, the effectof candidate compounds on expression of a cancer marker gene is assayedfor by detecting the level of cancer marker mRNA expressed by the cell.mRNA expression can be detected by any suitable method.

In other embodiments, the effect of candidate compounds on expression ofcancer marker genes is assayed by measuring the level of polypeptideencoded by the cancer markers. The level of polypeptide expressed can bemeasured using any suitable method, including but not limited to, thosedisclosed herein.

Specifically, the present disclosure provides screening methods foridentifying modulators, i.e., candidate or test compounds or agents(e.g., proteins, peptides, peptidomimetics, peptoids, small molecules orother drugs) which bind to gene fusions of the present disclosure, havean inhibitory (or stimulatory) effect on, for example, cancer markerexpression or cancer marker activity, or have a stimulatory orinhibitory effect on, for example, the expression or activity of acancer marker substrate. Compounds thus identified can be used tomodulate the activity of target gene products (e.g., cancer markergenes) either directly or indirectly in a therapeutic protocol, toelaborate the biological function of the target gene product, or toidentify compounds that disrupt normal target gene interactions.Compounds that inhibit the activity or expression of cancer markers areuseful in the treatment of proliferative disorders, e.g., cancer,particularly prostate, gastric or skin cancer.

In one embodiment, the disclosure provides assays for screeningcandidate or test compounds that are substrates of a cancer markerprotein or polypeptide or a biologically active portion thereof. Inanother embodiment, the disclosure provides assays for screeningcandidate or test compounds that bind to or modulate the activity of acancer marker protein or polypeptide or a biologically active portionthereof.

The test compounds of the present disclosure can be obtained using anyof the numerous approaches in combinatorial library methods known in theart, including biological libraries; peptoid libraries (libraries ofmolecules having the functionalities of peptides, but with a novel,non-peptide backbone, which are resistant to enzymatic degradation butwhich nevertheless remain bioactive; see, e.g., Zuckennann et al., J.Med. Chem. 37: 2678-85 [1994]); spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary and peptoid library approaches are preferred for use withpeptide libraries, while the other four approaches are applicable topeptide, non-peptide oligomer or small molecule libraries of compounds(Lam (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al., Proc. Natl. Acad. Sci.U.S.A. 90:6909 [1993]; Erb et al., Proc. Nad. Acad. Sci. USA 91:11422[1994]; Zuckermann et al., J. Med. Chem. 37:2678 [1994]; Cho et al.,Science 261:1303 [1993]; Carrell et al., Angew. Chem. Int. Ed. Engl.33.2059 [1994]; Carell et al., Angew. Chem. Int. Ed. Engl. 33:2061[1994]; and Gallop et al., J. Med. Chem. 37:1233 [1994].

Libraries of compounds may be presented in solution (e.g., Houghten,Biotechniques 13:412-421 [1992]), or on beads (Lam, Nature 354:82-84[1991]), chips (Fodor, Nature 364:555-556 [1993]), bacteria or spores(U.S. Pat. No. 5,223,409; herein incorporated by reference), plasmids(Cull et al., Proc. Nad. Acad. Sci. USA 89:18651869 [1992]) or on phage(Scott and Smith, Science 249:386-390 [1990]; Devlin Science 249:404-406[1990]; Cwirla et al., Proc. NatI. Acad. Sci. 87:6378-6382 [1990];Felici, J. Mol. Biol. 222:301 [1991]).

In one embodiment, an assay is a cell-based assay in which a cell thatexpresses a cancer marker mRNA or protein or biologically active portionthereof is contacted with a test compound, and the ability of the testcompound to the modulate cancer marker's activity is determined.Determining the ability of the test compound to modulate cancer markeractivity can be accomplished by monitoring, for example, changes inenzymatic activity, destruction or mRNA, or the like.

The ability of the test compound to modulate cancer marker binding to acompound, e.g., a cancer marker substrate or modulator, can also beevaluated. This can be accomplished, for example, by coupling thecompound, e.g., the substrate, with a radioisotope or enzymatic labelsuch that binding of the compound, e.g., the substrate, to a cancermarker can be determined by detecting the labeled compound, e.g.,substrate, in a complex.

Alternatively, the cancer marker is coupled with a radioisotope orenzymatic label to monitor the ability of a test compound to modulatecancer marker binding to a cancer marker substrate in a complex. Forexample, compounds (e.g., substrates) can be labeled with ¹²⁵I, ³⁵S ¹⁴Cor ³H, either directly or indirectly, and the radioisotope detected bydirect counting of radioemmission or by scintillation counting.Alternatively, compounds can be enzymatically labeled with, for example,horseradish peroxidase, alkaline phosphatase, or luciferase, and theenzymatic label detected by determination of conversion of anappropriate substrate to product.

The ability of a compound (e.g., a cancer marker substrate) to interactwith a cancer marker with or without the labeling of any of theinteractants can be evaluated. For example, a microphysiometer can beused to detect the interaction of a compound with a cancer markerwithout the labeling of either the compound or the cancer marker(McConnell et al. Science 257:1906-1912 [1992]). As used herein, a“microphysiometer” (e.g., Cytosensor) is an analytical instrument thatmeasures the rate at which a cell acidifies its environment using alight-addressable potentiometric sensor (LAPS). Changes in thisacidification rate can be used as an indicator of the interactionbetween a compound and cancer markers.

In yet another embodiment, a cell-free assay is provided in which acancer marker protein or biologically active portion thereof iscontacted with a test compound and the ability of the test compound tobind to the gene fusion protein, mRNA, or biologically active portionthereof is evaluated. Preferred biologically active portions of the genefusion proteins or mRNA to be used in assays of the present disclosureinclude fragments that participate in interactions with substrates orother proteins, e.g., fragments with high surface probability scores.

Cell-free assays involve preparing a reaction mixture of the target geneprotein and the test compound under conditions and for a time sufficientto allow the two components to interact and bind, thus forming a complexthat can be removed and/or detected.

In another embodiment, determining the ability of the cancer markerprotein or mRNA to bind to a target molecule can be accomplished usingreal-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolanderand Urbaniczky, Anal. Chem. 63:2338-2345 [1991] and Szabo et al. Curr.Opin. Struct. Biol. 5:699-705 [1995]). “Surface plasmon resonance” or“BIA” detects biospecific interactions in real time, without labelingany of the interactants (e.g., BIAcore). Changes in the mass at thebinding surface (indicative of a binding event) result in alterations ofthe refractive index of light near the surface (the optical phenomenonof surface plasmon resonance (SPR)), resulting in a detectable signalthat can be used as an indication of real-time reactions betweenbiological molecules.

In one embodiment, the target gene product or the test substance isanchored onto a solid phase. The target gene product/test compoundcomplexes anchored on the solid phase can be detected at the end of thereaction. Preferably, the target gene product can be anchored onto asolid surface, and the test compound, (which is not anchored), can belabeled, either directly or indirectly, with detectable labels discussedherein.

It may be desirable to immobilize cancer markers, an anti-cancer markerantibody or its target molecule to facilitate separation of complexedfrom non-complexed forms of one or both of the proteins, as well as toaccommodate automation of the assay. Binding of a test compound to acancer marker protein, or interaction of a cancer marker protein with atarget molecule in the presence and absence of a candidate compound, canbe accomplished in any vessel suitable for containing the reactants.Examples of such vessels include microtiter plates, test tubes, andmicro-centrifuge tubes. In one embodiment, a fusion protein can beprovided which adds a domain that allows one or both of the proteins tobe bound to a matrix. For example, glutathione-S-transferase-cancermarker fusion proteins or glutathione-S-transferase/target fusionproteins can be adsorbed onto glutathione Sepharose beads (SigmaChemical, St. Louis, Mo.) or glutathione-derivatized microtiter plates,which are then combined with the test compound or the test compound andeither the non-adsorbed target protein or cancer marker protein, and themixture incubated under conditions conducive for complex formation(e.g., at physiological conditions for salt and pH). Followingincubation, the beads or microtiter plate wells are washed to remove anyunbound components, the matrix immobilized in the case of beads, complexdetermined either directly or indirectly, for example, as describedabove.

Alternatively, the complexes can be dissociated from the matrix, and thelevel of cancer markers binding or activity determined using standardtechniques. Other techniques for immobilizing either cancer markersprotein or a target molecule on matrices include using conjugation ofbiotin and streptavidin. Biotinylated cancer marker protein or targetmolecules can be prepared from biotin-NHS (N-hydroxy-succinimide) usingtechniques known in the art (e.g., biotinylation kit, Pierce Chemicals,Rockford, EL), and immobilized in the wells of streptavidin-coated 96well plates (Pierce Chemical).

In order to conduct the assay, the non-immobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslynon-immobilized component is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe previously non-immobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the immobilized component (theantibody, in turn, can be directly labeled or indirectly labeled with,e.g., a labeled anti-IgG antibody).

This assay is performed utilizing antibodies reactive with cancer markerprotein or target molecules but which do not interfere with binding ofthe cancer markers protein to its target molecule. Such antibodies canbe derivatized to the wells of the plate, and unbound target or cancermarkers protein trapped in the wells by antibody conjugation. Methodsfor detecting such complexes, in addition to those described above forthe GST-immobilized complexes, include immunodetection of complexesusing antibodies reactive with the cancer marker protein or targetmolecule, as well as enzyme-linked assays which rely on detecting anenzymatic activity associated with the cancer marker protein or targetmolecule.

Alternatively, cell free assays can be conducted in a liquid phase. Insuch an assay, the reaction products are separated from unreactedcomponents, by any of a number of standard techniques, including, butnot limited to: differential centrifugation (see, for example, Rivas andMinton, Trends Biochem Sci 18:284-7 [1993]); chromatography (gelfiltration chromatography, ion-exchange chromatography); electrophoresis(see, e.g., Ausubel et al., eds. Current Protocols in Molecular Biology1999, J. Wiley: New York.); and immunoprecipitation (see, for example,Ausubel et al., eds. Current Protocols in Molecular Biology 1999, J.Wiley: New York). Such resins and chromatographic techniques are knownto one skilled in the art (See e.g., Heegaard J. Mol. Recognit 11:141-8[1998]; Hageand Tweed J. Chromatogr. Biomed. Sci. App 1 699:499-525[1997]). Further, fluorescence energy transfer may also be convenientlyutilized, as described herein, to detect binding without furtherpurification of the complex from solution.

The assay can include contacting the cancer markers protein, mRNA, orbiologically active portion thereof with a known compound that binds thecancer marker to form an assay mixture, contacting the assay mixturewith a test compound, and determining the ability of the test compoundto interact with a cancer marker protein or mRNA, wherein determiningthe ability of the test compound to interact with a cancer markerprotein or mRNA includes determining the ability of the test compound topreferentially bind to cancer markers or biologically active portionthereof, or to modulate the activity of a target molecule, as comparedto the known compound.

To the extent that cancer markers can, in vivo, interact with one ormore cellular or extracellular macromolecules, such as proteins,inhibitors of such an interaction are useful. A homogeneous assay can beused to identify inhibitors.

For example, a preformed complex of the target gene product and theinteractive cellular or extracellular binding partner product isprepared such that either the target gene products or their bindingpartners are labeled, but the signal generated by the label is quencheddue to complex formation (see, e.g., U.S. Pat. No. 4,109,496, hereinincorporated by reference, which utilizes this approach forimmunoassays). The addition of a test substance that competes with anddisplaces one of the species from the preformed complex will result inthe generation of a signal above background. In this way, testsubstances that disrupt target gene product-binding partner interactioncan be identified. Alternatively, gene fusion protein can be used as a“bait protein” in a two-hybrid assay or three-hybrid assay (see, e.g.,U.S. Pat. No. 5,283,317; Zervos et al., Cell 72:223-232 [1993]; Maduraet al., J. Biol. Chem. 268.12046-12054 [1993]; Bartel et al.,Biotechniques 14:920-924 [1993]; Iwabuchi et al., Oncogene 8:1693-1696[1993]; and Brent WO 94/10300; each of which is herein incorporated byreference), to identify other proteins, that bind to or interact withgene fusions and are involved in gene fusion activity. Such genefusion-bps can be activators or inhibitors of signals by the cancermarker proteins or targets as, for example, downstream elements of acancer markers-mediated signaling pathway.

Modulators of gene fusion expression can also be identified. Forexample, a cell or cell free mixture is contacted with a candidatecompound and the expression of cancer marker mRNA or protein evaluatedrelative to the level of expression of cancer marker mRNA or protein inthe absence of the candidate compound. When expression of cancer markermRNA or protein is greater in the presence of the candidate compoundthan in its absence, the candidate compound is identified as astimulator of cancer marker mRNA or protein expression. Alternatively,when expression of cancer marker mRNA or protein is less (i.e.,statistically significantly less) in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of cancer marker mRNA or protein expression. The level ofcancer markers mRNA or protein expression can be determined by methodsdescribed herein for detecting cancer markers mRNA or protein.

A modulating agent can be identified using a cell-based or a cell freeassay, and the ability of the agent to modulate the activity of a cancermarkers protein can be confirmed in vivo, e.g., in an animal such as ananimal model for a disease (e.g., an animal with prostate cancer ormetastatic prostate cancer; or an animal harboring a xenograft of aprostate cancer from an animal (e.g., human) or cells from a cancerresulting from metastasis of a prostate cancer (e.g., to a lymph node,bone, or liver), or cells from a prostate cancer cell line.

This disclosure further pertains to novel agents identified by theabove-described screening assays (see e.g., below description of cancertherapies). Accordingly, it is within the scope of this disclosure tofurther use an agent identified as described herein (e.g., a cancermarker modulating agent, an antisense cancer marker nucleic acidmolecule, a siRNA molecule, a cancer marker specific antibody, or acancer marker-binding partner) in an appropriate animal model (such asthose described herein) to determine the efficacy, toxicity, sideeffects, or mechanism of action, of treatment with such an agent.Furthermore, novel agents identified by the above-described screeningassays can be, e.g., used for treatments as described herein.

VI. Transgenic Animals

The present disclosure contemplates the generation of transgenic animalscomprising an exogenous cancer marker gene (e.g., gene fusion) of thepresent disclosure or mutants and variants thereof (e.g., truncations orsingle nucleotide polymorphisms). In preferred embodiments, thetransgenic animal displays an altered phenotype (e.g., increased ordecreased presence of markers) as compared to wild-type animals. Methodsfor analyzing the presence or absence of such phenotypes include but arenot limited to, those disclosed herein. In some preferred embodiments,the transgenic animals further display an increased or decreased growthof tumors or evidence of cancer.

The transgenic animals of the present disclosure find use in drug (e.g.,cancer therapy) screens. In some embodiments, test compounds (e.g., adrug that is suspected of being useful to treat cancer) and controlcompounds (e.g., a placebo) are administered to the transgenic animalsand the control animals and the effects evaluated.

The transgenic animals can be generated via a variety of methods. Insome embodiments, embryonal cells at various developmental stages areused to introduce transgenes for the production of transgenic animals.Different methods are used depending on the stage of development of theembryonal cell. The zygote is the best target for micro-injection. Inthe mouse, the male pronucleus reaches the size of approximately 20micrometers in diameter that allows reproducible injection of 1-2picoliters (pl) of DNA solution. The use of zygotes as a target for genetransfer has a major advantage in that in most cases the injected DNAwill be incorporated into the host genome before the first cleavage(Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438-4442 [1985]). As aconsequence, all cells of the transgenic non-human animal will carry theincorporated transgene. This will in general also be reflected in theefficient transmission of the transgene to offspring of the foundersince 50% of the germ cells will harbor the transgene. U.S. Pat. No.4,873,191 describes a method for the micro-injection of zygotes; thedisclosure of this patent is incorporated herein in its entirety.

In other embodiments, retroviral infection is used to introducetransgenes into a non-human animal. In some embodiments, the retroviralvector is utilized to transfect oocytes by injecting the retroviralvector into the perivitelline space of the oocyte (U.S. Pat. No.6,080,912, incorporated herein by reference). In other embodiments, thedeveloping non-human embryo can be cultured in vitro to the blastocyststage. During this time, the blastomeres can be targets for retroviralinfection (Janenich, Proc. Natl. Acad. Sci. USA 73:1260 [1976]).Efficient infection of the blastomeres is obtained by enzymatictreatment to remove the zona pellucida (Hogan et al., in Manipulatingthe Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. [1986]). The viral vector system used to introduce thetransgene is typically a replication-defective retrovirus carrying thetransgene (Jahner et al., Proc. Natl. Acad Sci. USA 82:6927 [1985]).Transfection is easily and efficiently obtained by culturing theblastomeres on a monolayer of virus-producing cells (Stewart, et al.,EMBO J., 6:383 [1987]). Alternatively, infection can be performed at alater stage. Virus or virus-producing cells can be injected into theblastocoele (Jahner et al., Nature 298:623 [1982]). Most of the founderswill be mosaic for the transgene since incorporation occurs only in asubset of cells that form the transgenic animal. Further, the foundermay contain various retroviral insertions of the transgene at differentpositions in the genome that generally will segregate in the offspring.In addition, it is also possible to introduce transgenes into thegermline, albeit with low efficiency, by intrauterine retroviralinfection of the midgestation embryo (Jahner et al., supra [1982]).Additional means of using retroviruses or retroviral vectors to createtransgenic animals known to the art involve the micro-injection ofretroviral particles or mitomycin C-treated cells producing retrovirusinto the perivitelline space of fertilized eggs or early embryos (PCTInternational Application WO 90/08832 [1990], and Haskell and Bowen,Mol. Reprod. Dev., 40:386 [1995]).

In other embodiments, the transgene is introduced into embryonic stemcells and the transfected stem cells are utilized to form an embryo. EScells are obtained by culturing pre-implantation embryos in vitro underappropriate conditions (Evans et al., Nature 292:154 [1981]; Bradley etal., Nature 309:255 [1984]; Gossler et al., Proc. Acad. Sci. USA 83:9065[1986]; and Robertson et al., Nature 322:445 [1986]). Transgenes can beefficiently introduced into the ES cells by DNA transfection by avariety of methods known to the art including calcium phosphateco-precipitation, protoplast or spheroplast fusion, lipofection andDEAE-dextran-mediated transfection. Transgenes may also be introducedinto ES cells by retrovirus-mediated transduction or by micro-injection.Such transfected ES cells can thereafter colonize an embryo followingtheir introduction into the blastocoel of a blastocyst-stage embryo andcontribute to the germ line of the resulting chimeric animal (forreview, See, Jaenisch, Science 240:1468 [1988]). Prior to theintroduction of transfected ES cells into the blastocoel, thetransfected ES cells may be subjected to various selection protocols toenrich for ES cells which have integrated the transgene assuming thatthe transgene provides a means for such selection. Alternatively, thepolymerase chain reaction may be used to screen for ES cells that haveintegrated the transgene. This technique obviates the need for growth ofthe transfected ES cells under appropriate selective conditions prior totransfer into the blastocoel.

In still other embodiments, homologous recombination is utilized toknock-out gene function or create deletion mutants (e.g., truncationmutants). Methods for homologous recombination are described in U.S.Pat. No. 5,614,396, incorporated herein by reference.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentdisclosure and are not to be construed as limiting the scope thereof.

Example 1 Materials and Methods

Samples and paired-end library preparation for Illumina sequencing.Prostate cancer tissues negative for ETS family gene rearrangements wereselected for paired-end sequencing from the University of Michigantissue core with informed consent from subjects. (SPORE in ProstateCancer (Tissue/Serum/Urine) Bank Institutional Review Board #1994-0481).Total RNA was isolated with Trizol (Invitrogen), according to themanufacturer's instructions. Quality assessment of RNA was performedwith the Agilent Bioanalyzer 2100 (Agilent Technologies). Paired-endlibraries (n=15) for sequencing with Illumina Genome Analyzer II wereprepared according to the protocol provided by Illumina, with minormodifications, with the mRNA-seq sample prep kit (Illumina). Sequenceanalysis was carried out by the Illumina data analysis pipeline.

Nomination of prostate gene fusions. Mate-pair transcriptome reads weremapped to the human genome (hg18) and Refseq transcripts, allowing up totwo mismatches, with the Illumina Genome Analyzer Pipeline softwareELAND (Efficient Alignment of Nucleotide Databases). Sequence alignmentswere subsequently processed to nominate gene fusions, using previouslydescribed methodology (Maher, C. A. et al. Nature 458, 97-101 (2009)).In brief, mate pairs were processed to identify any that eitherencompassed or spanned the fusion junction. Encompassing mate pairsrefer to those in which each read aligns to an independent transcript,thereby encompassing the fusion junction. In contrast, spanning matepairs refer to those in which one sequence read aligns to a gene and itsmate spans the fusion junction. Both categories undergo a series offiltering steps to remove putative false positives before being mergedtogether to generate the final chimera nominations.

Cloning of full-length fusion transcript. The full-length fusiontranscripts of SLC45A3-BRAF and ESRP1-RAF1 were cloned into pCR8/GW/TOPOEntry vector by TA cloning method (Invitrogen). All entry vector cloneswere sequence-confirmed and recombined into the Gateway pcDNA-DEST40mammalian expression vector (Invitrogen) and the pAd/CMV/V5-DESTAdenoviral expression system (Invitrogen) by LR Clonase II (Invitrogen).Plasmids with N terminus Flag and C terminus V5 tags were generated forinitial verification of protein expression in HEK293 cells.

Cell invasion and proliferation assays. Equal numbers of cells wereplated into 96-well plates, and a cell proliferation assay was performedusing WST-1 reagent (Roche) following the manufacturer's protocol. Forthe Boyden chamber Matrigel invasion assay, equal numbers of cells wereplated into each Matrigelcoated transwell in the presence of sorafenibor U0126 (Calbiochem) or DMSO (Sigma). Invasion assays were performed asdescribed previously (Kleer, C. G. et al. Proc. Natl. Acad. Sci. USA100, 11606-11611 (2003); Cao, Q. et al. Oncogene 27, 7274-7284 (2008)).

In vitro soft agar growth. For in vitro growth in soft agar, 2 ml of0.6% SeaPlaque GTG Agarose (Cambrex) dissolved in complete keratinocytesupplement-free medium (Invitrogen) was poured into six-well dishes.After polymerization, a second layer containing 2 ml of 0.4% agar incomplete keratinocyte supplement-free medium, and RWPE cells stablyexpressing SLC45A3-BRAF or ESRP1-RAF1 (1×10⁴ cells per well) were pouredon top. The next day, cells were treated with sorafenib or U0126 (10 μM)in 1 ml of supplement-free keratinocyte medium. Soft-agar assay plateswere incubated for 14 d at 37° C. MEK inhibitor (U0126) or sorafenib waschanged once a week. Each experimental condition was done in triplicate.On day 14, colonies larger than 40 μm in diameter were counted.

Fluorescence in situ hybridization. FISH hybridizations were performedon tissue microarrays of prostate, melanoma, gastric, endometrial andliver cancer types. Rearrangement-positive cases identified from tissuemicroarray were further validated on individual formalin-fixed andparaffin-embedded sections. Bacterial artificial chromosome clones wereselected from the University of California-Santa Cruz genome browser andpurchased through BACPAC resources (Children's Hospital, Oakland,Calif.). After colony purification, Midi-prep DNA was prepared withQiagenTips-100 (Qiagen). DNA was labeled by nick translation withbiotin-16-dUTP and digoxigenin-11-dUTP (Roche). Probe DNA wasprecipitated and dissolved in hybridization mixture containing 50%formamide, 2× saline sodium citrate, 10% dextran sulfate (ChemiconInternational) and 1% Denhardt's solution (Sigma). Approximately 200 ngof labeled probe was hybridized to normal human chromosomes to confirmthe map position of each BAC clone. FISH signals were obtained withdigoxigenin-fluorescein and Alexa Fluor 594 conjugates, to obtain greenand red colors, respectively. Fluorescence images were captured with ahigh-resolution charge-coupled device camera controlled by In SituImaging System image processing software (Metasystems).

MEK-ERK signaling pathway analysis. Stable pooled populations of RWPEcells expressing SLC45A3-BRAF or ESRP1-RAF1 were maintained insupplement-free medium without supplements for 2 h. For MEK inhibitortreatments, U0126 (10 μM) was added in the supplement-free keratinocytemedium for 2 h. MEK and ERK activation was assessed by western blotanalysis with antibodies to phospho-MEK or ERK and total MEK or ERKantibodies (Cell Signaling Technologies).

Statistical analyses. All data are presented as means±s.e.m., andsignificance was determined by two-tailed Student's t test.

Cell lines and Tissues. NIH3T3, RWPE-1, and HEK293 cell lines wereobtained from the American Type Culture Collection. Prostate cancertissues were obtained from the University of Michigan tissue core andUniversity of Michigan Rapid Autopsy Program which are part of theUniversity of Michigan Prostate Cancer Specialized Program Of ResearchExcellence (S.P.O.R.E). Five different tissue microarrays containing atotal of 512 samples with 90-100 samples each comprising samples forprostate cancer progression with benign prostate, PIN prostate, prostatetumors and warm autopsy samples. Each of the tissue microarrays wereused for the FISH evaluation of both RAF1 and BRAF. Prostate tissuesobtained from the radical prostatectomy series at the UniversityHospital Ulm (Ulm, Germany) comprised of 149 cases of primary prostatecancer (n=136) and lymph node metastasis (n=13) from patients thatunderwent radical prostatectomy and lymph node dissection between 1989and 2001 at Ulm University (Ulm, Germany) and were collected under anIRB approved protocol. The mean patient age was 64. The distribution ofGleason pattern was as follows: GS 6 (2%), GS 7 (24%), GS 8-10 (68%), GSN/A (6%). The pT staging distribution is as follows: pT2 (5%), pT3(93%), pT N/A (2%). The PSA levels range from 0.6-262 ng ml-1 (median23.75 ng ml-1). 25 (18%) patients received androgen-deprivatio therapypre-operatively. A tissue microarray was constructed using three 0.6 mmhigh density cancer tissue cores per case. A small cohort of 59transurethral resection of prostate (TURP) FFPE material was retrievedfrom McGill University Hospitals (Montreal, Canada). The patients hadbeen treated with one or multiple therapeutic protocols (radiationtherapy, brachytherapy and/or androgen deprivation therapy). Thecastration-resistant status was determined clinically based on PSAlevels and disease progression under treatment. Tissue cores atdiameters of 0.6 mm were obtained from areas containing high densitytumor and subjected to tissue microarray construction.

Real Time PCR validation. Quantitative PCR (QPCR) was performed usingPower SYBR Green Mastermix (Applied Biosystems) on an Applied BiosystemsStepOnePlus Real-Time PCR System. All oligonucleotide primers wereobtained from Integrated DNA Technologies and are listed below. TheGAPDH primer was used as a control. All assays were performed andrepeated twice and results were plotted as average fold change relativeto GAPDH.

SLC45A3 F (SEQ ID NO: 1) 5′-AGCCGCGCGCCTCGGCCA-3′ BRAF R (SEQ ID NO: 2)5′-ATCAGGAATCTCCCAATCATCACT-3′ SLC45A3 F (SEQ ID NO: 3)5′-GTACCAGCCCCACCCCTCTATCC-3′ SLC45A3 R (SEQ ID NO: 4)5′-TCAGTGGACAGGAAACGCACCATA-3′ BRAF EX8-Stop F (SEQ ID NO: 5)5′-GCCCCAAATTCTCACCAGTCCGTC-3′ BRAF EX8-Stop R (SEQ ID NO: 6)5′-TCAGTGGACAGGAAACGCACCA-3′ BRAF EX10-Stop F (SEQ ID NO: 7)5′-ATGAAACACTTGGTAGACGGGA-3′ BRAF EX10-Stop R (SEQ ID NO: 8)5′-TCAGTGGACAGGAAACGCACCA-3′ BRAF EX2 F (SEQ ID NO: 9)5′-AACATATAGAGGCCCTATTGGACA-3′ BRAF EX3 R (SEQ ID NO: 10)5′-AGAAGATGTAACGGTATCCATTG-3′ BRAF EX4 F (SEQ ID NO: 11)5′-GGAGTTACAGTCCGAGACAGTCTAA-3′ BRAF EX5 R (SEQ ID NO: 12)5′-CAGTAAGCCAGGAAATATCAGTGTC-3′ BRAF EX6 F (SEQ ID NO: 13)5′-AGCGTTGTAGTACAGAAGTTCCACT-3′ BRAF EX7 R (SEQ ID NO: 14)5′-AGATGTTAGGGCAGTCTCTGCTA-3′ BRAF EX8 F (SEQ ID NO: 15)5′-TGTGCATATAAACACAATAGAACCTG-3′ BRAF EX10 R (SEQ ID NO: 16)5′-TTCGATTCCTGTCTTCTGAGG-3′ BRAF EX11F (SEQ ID NO: 17)5′-AAAACACTTGGTAGACGGGACTC-3′ BRAF EX12R (SEQ ID NO: 18)5′-CTTGTAACTGCTGAGGTGTAGGTG-3′ BRAF EX13 F (SEQ ID NO: 19)5′-TTGTATCACCATCTCCATATCATTG-3′ BRAF EX14 R (SEQ ID NO: 20)5′-GGATGATTGACTTGGCGTGTA-3′ BRAF EX15 F (SEQ ID NO: 21)5′-CTACAGTGAAATCTCGATGGAGTG-3′ BRAF EX16 R (SEQ ID NO: 22)5′-TCATACAGAACAATTCCAAATGC-3′ BRAF EX17 F (SEQ ID NO: 23)5′-CGAGGATACCTGTCTCCAGAT-3′ BRAF EX18 R (SEQ ID NO: 24)5′-GATGCACTGCGGTGAATTTTT-3′ BRAF 3′UTR F (SEQ ID NO: 25)5′-AGTGAGAGAGTTCAGGAGAGTAGCA-3′ BRAF 3′UTR R (SEQ ID NO: 26)5′-AAGTATAAATTTTAGTTTGGGGAAAAA-3′ RAF1 EX5 F (SEQ ID NO: 27)5′-CATGAGCACTGTAGCACCAAA-3′ ESRP1 EX14 R (SEQ ID NO: 28)5′-AGCAGCTGTAGGGAAGTAGCC-3′ ESRP1 EX13 F (SEQ ID NO: 29)5′-GTACTACCCAGCAGGCACTCA-3′ RAF1 Ex6 R (SEQ ID NO: 30)5′-CTGGGACTCCACTATCACCAA-3′ RAF1 F (SEQ ID NO: 31)5′-ATGGAGCACATACAGGGAGCT-3′ ESRP1 R (SEQ ID NO: 32)5′-TTAAATACAAACCCATTCTTTGG-3′ ESRP1 F (SEQ ID NO: 33)5′-ATGACGGCCTCTCCGGATTA-3′ RAF1 R (SEQ ID NO: 34)5′-CTAGAAGACAGGCAGCCTCG-3′ DUSP6 F (SEQ ID NO: 35)5′-CCGCAGGAGCTATACGAGTC-3′ DUSP6 R (SEQ ID NO: 36)5′-CCTCGTCCTTGAGCTTCTTG-3′ SPRY2 F (SEQ ID NO: 37)5′-CCCCTCTGTCCAGATCCATA-3′ SPRY2 R (SEQ ID NO: 38)5′-CCCAAATCTTCCTTGCTCAG-3′ AGTRAP F (SEQ ID NO: 39)5′-ATCCCTTTGCAGTCCCAGA-3′ BRAF R (SEQ ID NO: 40)5′-CTGTGGAATTGGAATGGATTTT-3′ GAPDH F (SEQ ID NO: 41)5′-TGCACCACCAACTGCTTAGC-3′ GAPDH R (SEQ ID NO: 42)5′-GGCATGGACTGTGGTCATGAG-3′

Gene Expression Profiling. LNCaP and VCaP cells were starved for 48hours and treated with 1 nM R1881 for 24 and 48 hours and RNA isolatedfrom these cells were used for microarray analysis. Gene expressionmicroarray profiling was performed using the Agilent Whole Human GenomeOligo Microarray according to the manufacturer's protocol.

Confirmation of SLC45A3-BRAF and ESRP1-RAF1 protein expression byWestern Blotting. The ESRP1-RAF1 fusion positive prostate cancer tissueand fusion negative tissues were homogenized in NP40 lysis buffer (50 mMTris-HCl, 1% NP40, pH 7.4, Sigma, St. Louis, Mo.), and complete proteaseinhibitor mixture (Roche) and phosphatase inhibitor (EMD Bioscience).Fresh frozen material for the SLC45A3-BRAF index case was not availablefor similar assay. For evaluating the expression and to assess themolecular weight of the fusion protein in the fusion positive tissues,HEK293 cells were separately transfected with SLC45A3-BRAF andESRP1-RAF1 fusion constructs (cloned in pDEST40 expressionvector—Invitrogen). A vector control and the transfected cells werelysed in NP40 lysis buffer with protease inhibitor. Fifteen microgramsof each protein extract were boiled in sample buffer, separated bySDS-PAGE, and transferred onto Polyvinylidene Difluoride membrane (GEHealthcare). The membrane was incubated for one hour in blocking buffer(Tris-buffered saline, 0.1% Tween (TBS-T), 5% nonfat dry milk) andincubated overnight at 4° C. with anti-BRAF (Santa Cruz) and anti-RAFTmouse monoclonal antibody (1:1000 in blocking buffer (BD Bioscience).Following three washes with TBS-T, the blot was incubated withhorseradish peroxidase-conjugated secondary antibody and the signalsvisualized by enhanced chemiluminescence system as described by themanufacturer (GE Healthcare). Blot was reprobed with anti-actin mousemonoclonal (1:5000, Sigma) antibodies.

Foci Formation Assay. NIH3T3 cells (1.5×10⁵) in 35-mm plastic disheswere transfected with 2 μg of DNA of the plasmid of interest. All thetransfections were performed using Fugene 6 according to themanufacturer's protocol (Roche Applied Sciences). Plasmids for fusiontranscripts SLC45A3-BRAF, BRAF Ex8-stop, and BRAF Ex10-stop and BRAFmutant V600E were used along with control plasmids (pDEST40 and pBABErespectively). Three days after transfection, cells were split into140-mm dishes containing DMEM with 5% Calf Serum (Life Technologies).The cultures were fed every 3-4 days. After 3 weeks, the cells werestained with 0.2% crystal violet in 70% ethanol for the visualization offoci, and were counted on colony counter (Oxford Optronix, softwarev4.1, 2003). Foci counts were further confirmed manually.

BRAFV⁶⁰⁰ Mutation Detection by Pyrosequencing. One to 2 μg of total RNAisolated from fresh frozen localized prostate cancer (n=229), metastaticprostate cancer (n=37) and benign prostate (n=8) tissue samples, and apanel of melanoma (n=34), gastric cancer (n=25) were converted into cDNAusing Superscript II Reverse Transcriptase (Invitrogen) according tomanufacturer's instructions. Biotinylated sequencing templates weregenerated by PCR amplification of a 375 bp fragment spanning themutation in codon 600 (V600, Exon 15) of the BRAF gene using primersfrom PyroMark Q24 BRAF kit (Biotage-Qiagen) according to manufacturer'sinstructions. Ten pl of the biotinylated PCR products were immobilizedon streptavidin coated Sepharose beads (Streptavidin Sepharose HighPerformance, GE Healthcare) using Pyromark Q24 Vacuum Prep Workstation,followed by removal of non-biotinylated strand by sodium hydroxidedenaturation followed by wash in neutralization buffer and 70% ethanol.The single stranded biotinylated templates were then mixed with 0.3 mMsequencing primer and ‘sequencing by synthesis’ was carried out throughdispensation of the query nucleotide sequence using PyroMark Q24platform, as described before. The nucleotide sequence ACAGA/TGAAA (SEQID NO:43) for codon 600 was analyzed and visualized by Pyromark Q241.0.10 software. A panel of 9 melanoma cell lines (SK-MEL-2, SK-MEL-5,SK-MEL-19, SK-MEL-28, SK-MEL-29, SK-MEL-103, G-361, Malme-3M, mel-1 withknown mutation status was used to serve as assay standards.

NIH3T3-SLC45A3-BRAF or RWPE-SLC45A3-BRAF Xenograft Models. Four week oldmale Balb C nu/nu mice were purchased from Charles River, Inc. (CharlesRiver Laboratory, Wilmington, Mass.). Stable polyclonal NIH3T3 cells orRWPE over-expressing fusion transcript SLC45A3-BRAF or vector pDEST40 orsingle clone (5×10⁶ cells) were resuspended in 100 pl of saline with 20%Matrigel (BD Biosciences). Cells were implanted subcutaneously into theleft flank region of the mice. Mice were anesthetized using a cocktailof xylazine (80-120 mg kg-1 IP) and ketamine (10 mg kg-1 IP) forchemical restraint before implantation. Ten mice were included in eachgroup. Growth in tumor volume was recorded weekly by using digitalcalipers and tumor volumes were calculated using the formula (π/6)(L×W²), where “L”=length of the tumor and “W”=width. All proceduresinvolving mice were approved by the University Committee on Use and Careof Animals (UCUCA) at the University of Michigan and conform to theirrelevant regulatory standards.

Results

To search for druggable rearrangements in prostate cancer, paired-end,massively parallel transcriptome sequencing was used to prioritizecandidate gene fusions in prostate tumors by generating a score derivedfrom the quantity of mate-pair reads that meet a series of computationalfilters implemented to reduce potential false-positive chimeranominations (Maher et al. Proc. Natl. Acad. Sci. USA 106, 12353-12358(2009)). Prioritization histograms for two ETS rearrangement—positiveprostate cancers, PCA1 and PCA2, which harbor AX747630 (Homo sapienscDNA FLJ35294 fis, clone PROST2008724)-ETV1 and TMPRSS2-ERG(transmembrane protease, serine 2-v-ets erythroblastosis virus E26oncogene homolog (avian)) gene fusions, respectively, indicate that theETS gene fusion had the highest score in each sample (FIG. 1a ), asreported previously (Maher et al., 2009, supra; Wang et al., Nat.Biotechnol. 27, 1005-1011 (2009)).

In this study, five ETS gene fusion-positive and ten ETS genefusion-negative prostate cancers (ETS gene fusion status was determinedby fluorescence in situ hybridization (FISH), quantitative RT-PCR(qRT-PCR) or both) were sequenced and it was found that two ETS-negativesamples, PCA3 and PCA17, each prioritized a fusion involving BRAF andRAF1 genes, key serine-threonine kinase components of the RAF signalingpathway (FIG. 1a ). Whereas activating somatic mutations in the RAFkinase pathway, such as BRAF with a mutation that results in a V600Eamino acid substitution (BRAFV600E), are common in melanoma, thyroid,colon and ovarian cancers (Cohen et al., J. Natl. Cancer Inst. 95,625-627 (2003); Davies et al., Nature 417, 949-954 (2002); Wang et al.,Cancer Res. 63, 5209-5212 (2003); Xing, Endocr. Relat. Cancer 12,245-262 (2005)), activating gene fusions of pathway members have beenreported less frequently and are found in subsets of relatively rarecancers (Ciampi et al. J. Clin. Invest. 115, 94-101 (2005); Jones et al.Cancer Res. 68, 8673-8677 (2008); Dessars et al. J. Invest. Dermatol.127, 1468-1470 (2007)). The RAF kinase pathway is druggable, withmultiple approved and investigational agents in late-stage development.Sorafenib, a US Food and Drug Administration-approved drug, wasoriginally identified as a RAF kinase inhibitor but was subsequentlyfound to target other kinases such as vascular endothelial growth factorreceptor-2 (VEGFR-2), VEGFR-3 and platelet-derived growth factorreceptor-β (Wilhelm et al. Mol. CancerTher. 7, 3129-3140 (2008)). Anemerging lead drug candidate, PLX-4032, is highly selective for theBRAFV600E mutation and is being evaluated in people with advancedmelanoma (Sala et al. Mol. Cancer Res. 6, 751-759 (2008)). Thus, thedruggable gene fusions identified in prostate tumors PCA3 and PCA17 werecharacterized.

The first case, PCA3, revealed an interchromosomal rearrangementresulting in the fusion of untranslated exon 1 of SLC45A3 with exon 8 ofBRAF (FIG. 1b ). SLC45A3 is a prostate-specific, androgen responsivegene that has been found fused to ERG (Esgueva et al. Mod. Pathol. 23,539-546 (2010); Han et al. Cancer Res. 68, 7629-7637 (2008)) ETV118,ETV519 and ELK4 (Maher et al. Nature 458, 97-101 (2009); Rickman et al.Cancer Res. 69, 2734-2738 (2009)) in a subset of prostate tumors. Thepredicted open reading frame encodes 329 amino acids of the C-terminalportion of BRAF (FIG. 5a ), retaining the kinase domain but losing theN-terminal RAS-binding domain, indicating that the mutant protein isconstitutively active. Having inherited promoter regulatory elementsfrom SLC45A3, this BRAF fusion is likely under androgen regulation (FIG.6). Consistent with this, the C-terminal exons of BRAF (8-18) present inthe fusion protein are overexpressed in PCA3 tumor relative to normalprostate and other prostate cancers (FIG. 7a,b ). The second case,PCA17, revealed two highly expressed gene fusions involving ESRP1 andRAF1 (FIG. 1c,d ), formed by a balanced reciprocal translocation. ESRP1is a splicing factor that regulates the formation of epithelialcell-specific isoforms of mRNA (Warzecha et al., Mol. Cell 33, 591-601(2009)), whereas RAF1 (or CRAF) is a serine-threonine protein kinase.

The ESRP1-RAF1 fusion transcript involves the fusion of exon 13 of ESRP1to exon 6 of RAF1 (FIG. 1c ). The predicted open reading frame encodes a120-kDa fusion protein comprised of the majority of ESRP1, including itsthree RNA recognition motifs, fused to the C-terminal kinase domain ofRAFT (FIG. 6c ). Loss of the RAS-binding domain of RAFT indicates thatthis fusion protein is constitutively active.

In addition to ESRP1-RAF1, the reciprocal gene fusion RAF1-ESRP1,produced from the same genomic rearrangement in PCA17, was alsodetected. The RAF1-ESRP1 transcript involves the fusion of exon 5 ofRAF1 with exon 14 of ESRP1 (FIG. 1d ), which encodes a predicted 30-kDaprotein comprised of the RAS-binding domain of RAF1 fused to 194 aminoacids from the C terminus of ESRP1 (FIG. 5c ). Unlike SLC45A3-BRAF,ESRP1-RAF1 is predicted to not be regulated by androgen, as wild-typeESRP1 is not androgen regulated (FIG. 6).

Next, the SLC45A3-BRAF fusion was confirmed by fusion-specific qPCR inPCA3 tumor. Rearrangement at the DNA level was validated by FISH andconfirmed the presence of two copies of rearranged chromosomes bybreak-apart (FIG. 8a ) and fusion (FIG. 2) assays. Expression of theSLC45A3-BRAF fusion gene in HEK293 cells and stable expression in RWPE(human benign immortalized prostate epithelial cell line) cellsgenerated a 37-kDa protein (FIG. 9a,b ).

ESRP1-RAF1 and RAF1-ESRP1 were also validated by qRTPCR (FIG. 2b ) inthe index PCA17 tumor. FISH confirmed the DNAlevel rearrangement andfusion of the ESRP1 and RAF1 loci (FIG. 2d and FIG. 8b ). Expression ofa 120-kDa ESRP1-RAFT fusion protein was observed in PCA17 tumor and uponoverexpression in HEK293 (FIG. 2g ) and RWPE cells (FIG. 9c ).

BRAF and RAF1 rearrangement frequencies were estimated in threeindependent prostate cancer clinical cohorts by FISH on tissuemicroarrays with break-apart probes. Out of 349 prostate cancer casesthat were evaluable by FISH, six cases had an aberration at the BRAFlocus (five rearrangements and one deletion of the 5′ probe), and fourof 450 cases showed rearrangement at the RAF1 locus (one rearrangementand three deletions of the 3′ probe). Other than the index cases PCA3and PCA17, these cases did not show rearrangement of the SLC45A3 orESRP1 loci, indicating fusions involving multiple 5′ partners, similarto ETV1 fusions in prostate cancer (Tomlins et al. Nature 448, 595-599(2007)). A majority of the cases that were positive for rearrangementsof BRAF or RAF1 had advanced features including high Gleason score andcastration resistance. All of the cases were negative for ETS generearrangement (except MET37, which had an ERG rearrangement), indicatingthat these aberrations occur predominantly in ETS-negative prostatecancers (Table 1a).

The analysis of BRAF and RAF1 rearrangements was extended to other solidtumors by using break-apart FISH probes on tissue microarrays of breast(n=49), endometrial (n=26), gastric (n=85), melanoma (n=131) and liver(n=42) tumors. Similar to the case in prostate cancer, a 1-2% incidenceof BRAF aberrations was found in gastric cancer (2 of 105) (FIG. 2e )and one case each of BRAF and RAF1 rearrangement in melanoma (2 of 131)(FIG. 2f ). In the gastric cancer index case GCT-15, paired-endtranscriptome sequencing revealed that exon 8 of the BRAF gene was fusedwith exon 5 of AGTRAP (encoding angiotensin II, type Ireceptor-associated protein) (FIG. 1e ). The AGTRAPBRAF fusiontranscript was validated by qRT-PCR (FIG. 2c ) and the DNA-levelrearrangement by FISH analysis (FIG. 2f ). The AGTRAP-BRAF fusionresulted in the formation of a 597-amino acid fusion protein with theC-terminal kinase domain of BRAF fused to the N-terminal angiotensin II,type 1 receptor-associated domain of AGTRAP (FIG. 5d ). The expressionof the predicted AGTRAP-BRAF fusion protein was confirmed by immunoblotanalysis of the index tumor GCT-15 (FIG. 2h ).

Considering the prevalence of oncogenic mutations in BRAF in variouscancer types, the BRAFV600E mutation was screened for by pyrosequencingin 274 prostate samples, 23 gastric cancer samples, two gastroesophagealcancer samples and 34 melanoma samples. It was found that 20 of 34 (59%)melanoma samples, one of 25 gastroesophageal cancers and zero of 274prostate samples were positive for the BRAFV600E mutation. The presentdisclosure is not limited to a particular mechanism. Indeed, anunderstanding of the mechanism is not necessary to practice the presentdisclosure. Nonetheless, none of the RAF pathway generearrangement-positive prostate cancers, gastroesophageal cancers andmelanomas identified herein harbored BRAFVa1600E mutations, indicatinggenomic rearrangement, rather than mutation, as a mechanism for RAF geneactivation in a subset of solid tumors. In an Asian cohort, 10% ofprostate cancer cases have been reported to be positive for BRAFV600Emutations (Cho et al. Int. J. Cancer 119, 1858-1862 (2006)). NoBRAFVal600 mutations were found in the prostate cancer cohorts, which isconsistent with a recently published study (zero of 95 prostate cancerswere positive for Val600) (MacConaill et al. PLoS One 4, e7887 (2009)).

The functional relevance of the fusions involving RAF pathway members inprostate cancer was examined. First, the SLC45A3-BRAF fusion wasexamined in mouse fibroblast NIH3T3 cells, a system classically used tostudy RAS and RAF biology (Garte et al., Cancer Res. 47, 3159-3162(1987)). Overexpression of SLC45A3-BRAF (FIG. 5b ) or mutant BRAFV600Eshowed a marked increase in the number of foci as compared to vectorcontrols (FIG. 3a ). The foci assay data were further validated byautomated colony counting (FIG. 10a ). NIH3T3 cells overexpressingSLC45A3-BRAF formed rapidly growing tumors in nude mice (FIG. 3b );however, NIH3T3 cells overexpressing ESRP1-RAF1 did not form tumors.

To examine the role of these fusions in the prostate, SLC45A3-BRAF orESRP1-RAF1 was overexpressed in RWPE cells; both gene fusions resultedin increased cell proliferation that was sensitive to the RAF kinaseinhibitor sorafenib (FIG. 3c,d ). A marked increase in cell invasion ofRWPE cells expressing either SLC45A3-BRAF or ESRP1-RAF1, which wassensitive to sorafenib or the MEK inhibitor U0126 was also observed(FIG. 4a,b ). Furthermore, RWPE cells expressing either SLC45A3-BRAF orESRP1-RAF1 formed anchorage independent colonies in soft agar, whichwere again sensitive to RAF and MEK inhibitors (FIG. 4c,d ). Finally,RWPE cells stably expressing SLC45A3-BRAF formed small tumors inimmunodeficient mice that regressed after 4 weeks (FIG. 10b ).

The RAF family is known to have a pivotal role in transducing signalsfrom RAS to downstream kinases, mitogen-activated protein kinase andextracellular signal-regulated kinase (ERK) kinase (MEK1/2) and ERK1/2(Hoeflich et al. Clin. Cancer Res. 15, 4649-4664 (2009)). Overexpressionof SLC45A3-BRAF or ESRP1-RAF1 in RWPE cells induced MEK and ERKphosphorylation, which was sensitive to treatment with a MEK inhibitor(FIG. 4e ). The MEK inhibitor also decreased MEK1/2 and ERK1/2phosphorylation in a control BRAFV600E mutation-positive human melanomacell line, SK-MEL-94, consistent with previous data (Pratilas et atProc. Natl. Acad. Sci. USA 106, 4519-4524 (2009). It was also found thatan increase in mRNA expression of genes encoding the feedback effectorsdual-specificity phosphatase-6 and sprouty homolog-2 in RWPE cellsstably expressing SLC45A3-BRAF or ESRP1-RAF1, and the expression ofthese feedback effectors was decreased upon MEK inhibitor treatment(FIG. 11).

The results emphasize the key role of the RAF pathway in prostate cancerdevelopment and progression. Although it is rare in human prostatetumors, activation of the BRAF pathway via the V600E mutation ingenetically engineered mice has been shown to cooperate with otherlesions to initiate the development of invasive prostate cancer (Jeonget al. PLoS One 3, e3949 (2008). ETS transcription factors, includingETV1, have been shown to be downstream targets activated by theRAS-RAF-MAPK signaling pathway (Janknecht, Mol. Cell. Biol. 16,1550-1556 (1996); Bosc et al., J. Cell. Biochem. 86, 174-183 (2002)).

Sequencing tumor transcriptomes and genomes identifies rare targetablefusions across cancer types. Screening for RAF kinase fusions is usefulin identifying people with cancer who benefit from treatment with RAFkinase inhibitors. The identification of RAF pathway gene rearrangementsin 1-2% of prostate cancers, gastric cancers and melanomas supports thegeneral principle that cancers should be classified by driving molecularevents, rather than by organ site, in the context of rational targetedtherapy.

TABLE 1 Clinicopathological characteristics of the index cases inprostate, gastric cancer and melanoma with BRAF and RAF1 generearrangement a Sample Gleason ERG BRAF RAF1 ID Age Diagnosis Score CRrearrangement rearrangement rearrangement Fusion BRAF PCA3 59 PCA 4 + 4− − + − SLC45A3- BRAF PCA44 75 PCA 4 + 4 + − + − PCA45 NA PCA 4 + 4 +− + − PCA46 89 PCA 5 + 4 + − + − MET37 63 MET 4 + 5 + + + − PCA47 62 PCA4 + 3 − − 5’del − RAF1 PCA17 NA PCA 3 + 4 − − − + ESRP1- RAF1 PCA48 66PCA 3 + 3 − − − 3’del MET36 62 MET NA + − − 3’del PCA49 NA PCA NA − − −3’del b Sample BRAF RAF1 ID Age Sex Diagnosis rearrangementRearrangement Fusion GCT15 61 F Gastric + − AGTRAP- adenocarcinoma BRAFGC#10 51 F Adenocarcinoma 5’del − − of GE junction MEL23 58 MMetastatic + − − Melanoma MEL24 88 F Metastatic − + − Melanoma CR:Castration-resistant: PCA, clinically localized prostate cancer; MET,metastatic prostate cancer

Although a variety of embodiments have been described in connection withthe present disclosure, it should be understood that the claimedinvention should not be unduly limited to such specific embodiments.Indeed, various modifications and variations of the describedcompositions and methods of the invention will be apparent to those ofordinary skill in the art and are intended to be within the scope of thefollowing claims.

We claim:
 1. A composition comprising an oligonucleotide probe thathybridizes to a junction of a chimeric genomic DNA or a chimeric mRNA,wherein a 5′ portion of the chimeric genomic DNA or the chimeric mRNA isfrom an SLC45A3gene and a 3′ portion of the chimeric genomic DNA or thechimeric mRNA is from a RAF family member gene.
 2. The composition ofclaim 1 wherein the RAF family member gene is BRAF.
 3. The compositionof claim 1 wherein the 3′ portion of the chimeric genomic DNA or thechimeric mRNA comprises a wild-type sequence of the RAF family membergene.
 4. The composition of claim 1 further comprising a chimericgenomic DNA or a chimeric mRNA, wherein a 5′ portion of the chimericgenomic DNA or the chimeric mRNA is from an SLC45A3 gene and a 3′portion of the chimeric genomic DNA or the chimeric mRNA is from a RAFfamily member gene.
 5. The composition of claim 1 wherein the 3′ portionof the chimeric genomic DNA or the chimeric mRNA comprises exon 8 ofBRAF.
 6. The composition of claim 1 wherein the chimeric genomic DNA orthe chimeric mRNA comprises exon 1 of SLC45A3 fused to exon 8 of BRAF.7. The composition of claim 1 wherein the oligonucleotide probe islabeled.
 8. The composition of claim 4 wherein the oligonucleotide probeis hybridized to said chimeric genomic DNA or said chimeric mRNA.